CN108064296A - SPT6 nucleic acid molecule for controlling insect pests - Google Patents

Abstract

The present disclosure relates to nucleic acid molecules and methods of using the same for controlling insect pests by RNA interference-mediated inhibition of target coding sequences and transcribed non-coding sequences in insect pests, including coleopteran pests. The disclosure also relates to methods for making transgenic plants expressing nucleic acid molecules useful for controlling insect pests, and plant cells and plants obtained thereby.

Description

SPT6 nucleic acid molecule for controlling insect pests

priority statement

This application claims the benefit of filing of US Provisional Patent Application Serial No. 62/168,606, "SPT6NUCLEIC ACID MOLECULES TO CONTROL INSECT PESTS," filed May 29, 2015.

technical field

The present invention generally relates to the genetic control of plant damage caused by insect pests, such as coleopteran pests. In particular embodiments, the present invention relates to the identification of target-encoding polynucleotides and non-coding polynucleotides, and the post-transcriptional repression or inhibition of target-encoding and non-coding polynucleotides in insect pest cells using recombinant DNA techniques. expression, thereby providing plant protection effects.

Background technique

Western corn rootworm (WCR) (Diabrotica virgifera virgifera LeConte) is one of the most destructive corn rootworm species in North America and is of great concern in the corn growing regions of the Midwestern United States. Northern corn rootworm (NCR) (Diabrotica barberi Smith and Lawrence) is a closely related species commensal with WCR in almost the same range. Several other related subspecies of Diabrotica are serious pests in the Americas: Mexican corn rootworm (MCR) (D. virgifera zeae Krysan and Smith); southern corn rootworm ( SCR) (D. undecimpunctata howardi Barber); Cucumber root beetle (D. balteata LeConte); Cucumber eleven-star beetle (D. undecimpunctata tenella); South American leaf beetle (D. .speciosa Germar); and D.u.undecimpunctata Mannerheim. The USDA has estimated that corn rootworm causes $1 billion in lost revenue annually, including $800 million in lost yield and $200 million in treatment costs.

Both WCR eggs and NCR eggs are deposited in the soil during the summer. These insects remain in the egg stage throughout the winter. Eggs are oval, white, and less than 0.004 inches long. The larvae hatch in late May or early June, with the precise timing of egg hatching varying from year to year due to temperature differences and location. Newly hatched larvae are white worms that are less than 0.125 inches long. Once hatched, the larvae begin feeding on corn roots. Corn rootworms go through three larval instars. After several weeks of feeding, the larvae molt and enter the pupal stage. They pupate in the soil and then emerge from the soil as adults in July and August. Adult rootworms are about 0.25 inches in length.

Corn rootworm larvae complete development on corn and several other grass species. Larvae reared on yellow foxtail emerged later and adults had smaller headshell sizes than larvae reared on maize. Ellsbury et al. (2005) Environ. Entomol. 34:627-34. WCR adults feed on corn silk, pollen, and corn seeds on exposed ear tips. If WCR adults emerge before maize reproductive tissues are present, they may feed on leaf tissue, slowing plant growth and occasionally killing the host plant. However, once there is a preferred silk and pollen, adults quickly move to it. NCR adults also feed on the reproductive tissue of maize plants, but rarely on maize leaves.

Most rootworm damage in corn is caused by larval feeding. Newly hatched rootworms initially feed on the delicate corn root hairs, burrowing into the root tips. As the larvae grow larger, they feed on and burrow into primary roots. When corn rootworms are present in large numbers, larval feeding often results in root pruning down to the base of the stalk. Severe root damage impairs the ability of the roots to transport water and nutrients into the plant, slows plant growth, and leads to reduced grain production, often drastically reducing overall yield. Severe root damage also often leads to corn plant lodging, which makes harvesting more difficult and further reduces yields. Additionally, adult feeding on maize reproductive tissue can lead to silk pruning at the ear tip. If this "silk shearing" is severe enough during pollen shedding, pollination may be disrupted.

Attempts to control maize can be achieved through crop rotation, chemical insecticides, biological insecticides (e.g., the spore-forming Gram-positive bacterium Bacillus thuringiensis), transgenic plants expressing Bt toxin, or combinations of these means rootworm. The downside of crop rotation is that it unnecessarily limits the uses of farmland. Additionally, some rootworm species may lay eggs in soybean fields, reducing the efficiency of crop rotations with corn and soybeans.

Chemical insecticides are the most heavily relied upon strategy for achieving corn rootworm control. Nonetheless, the use of chemical insecticides is not a perfect corn rootworm control strategy; if the cost of chemical insecticides is added to the losses due to rootworm damage Corn rootworm could cost more than $1 billion. Large larval populations, heavy rainfall, and poor pesticide application can all lead to inadequate corn rootworm control. In addition, continued use of insecticides may select for insecticide-resistant rootworm species and risks serious environmental impacts due to insecticide toxicity to non-target species.

RNA interference (RNAi) is a method that exploits endogenous cellular pathways whereby interfering RNA (iRNA) molecules (such as dsRNA molecules) specific for the entire target gene or any portion of the target gene of sufficient size Causes degradation of the mRNA encoded by it. In recent years, RNAi has been used to perform gene "knockdown" in cells in many species and experimental systems such as Caenorhabditis elegans, plants, insect embryos and tissue culture. See eg Fire et al., (1998) Nature 391:806-11; Martinez et al., (2002) Cell 110:563-74; McManus and Sharp, (2002) Nature Rev. Genetics 3:737-47.

RNAi achieves mRNA degradation through intrinsic pathways including the DICER protein complex. DICER cuts long dsRNA molecules into short fragments of about 20 nucleotides, called small interfering RNAs (siRNAs). The siRNA unwinds into two single-stranded RNAs: a passenger strand and a guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). MicroRNAs (miRNAs), which are structurally very similar molecules cleaved from precursor molecules containing a polynucleotide "loop" connected to hybridized passenger and guide strands, can be similarly incorporated into RISC . Post-transcriptional gene silencing occurs when the guide strand specifically binds to a complementary mRNA molecule and induces cleavage by Argonaute, the catalytic component of the RISC complex. Although in some eukaryotes such as plants, nematodes and some insects the initial concentration of siRNA and/or miRNA is limited, this process is known to be systematic throughout the organism.

Only transcripts complementary to siRNAs and/or miRNAs are cleaved and degraded, thus knockdown of mRNA expression is sequence-specific. In plants, there are several functional groups of DICER genes. The gene-silencing effects of RNAi last for days and, under experimental conditions, can lead to a 90% or more reduction in the abundance of targeted transcripts, with consequent reductions in the levels of the corresponding proteins. In insects, there are at least two DICER genes, among which DICER1 promotes the degradation of miRNAs under the guidance of Argonaute1. Lee et al., (2004) Cell 117(1):69-81. DICER2 promotes the degradation of siRNA under the guidance of Argonaute2.

US Patent No. 7,612,194 and US Patent Publication Nos. 2007/0050860, 2010/0192265, and 2011/0154545 disclose libraries of 9112 expressed sequence tag (EST) sequences isolated from pupae of Dvvirgifera LeConte. Nucleic acid molecules that will be complementary to one of several specific partial sequences of the vacuolar H ⁺ -ATPase (V-ATPase) of the corn root firefly beetle disclosed therein are proposed in U.S. Patent No. 7,612,194 and U.S. Patent Publication No. 2007/0050860 Operably linked to a promoter to express the antisense RNA in plant cells. U.S. Patent Publication No. 2010/0192265 proposes to express antisense RNA in plant cells by operably linking a promoter to a nucleic acid molecule that is related to the gene of the corn root firefly beetle with unknown and undisclosed function A specific partial sequence is complementary (this partial sequence is said to be 58% identical to the C56C10.3 gene product in C. elegans). U.S. Patent Publication No. 2011/0154545 proposes the expression of antisense RNA in plant cells by operably linking a promoter to a nucleic acid molecule that is identical to the coatomer beta subunit gene Two specific parts of the sequence are complementary. In addition, U.S. Patent No. 7,943,819 discloses a library of 906 expressed sequence tag (EST) sequences isolated from corn root firefly beetle larvae, pupae, and dissected midgut, and proposes to operably link the promoter to A nucleic acid molecule for expressing double-stranded RNA in plant cells, the nucleic acid molecule is complementary to a specific partial sequence of the charged multivesicular body protein 4b gene of the corn root firefly beetle.

In addition to several specific partial sequences of V-ATPase and specific partial sequences of genes with unknown functions, there is no further suggestion in U.S. Patent No. 7,612,194 and U.S. Patent Publication Nos. Any specific sequence from the over 9,000 listed sequences for RNA interference. Furthermore, neither U.S. Patent No. 7,612,194 nor U.S. Patent Publication Nos. 2007/0050860, 2010/0192265, and 2011/0154545 teach which of the more than 9,000 sequences they provide additional sequences are useful in corn rootworm species when used as dsRNA or siRNA can be deadly, and it's not even taught to be useful in any other way. Apart from the specific partial sequence of the charged multivesicular body protein 4b gene, US Patent No. 7,943,819 does not suggest the use of any specific sequence of the over 900 sequences listed therein for RNA interference. Furthermore, US Patent No. 7,943,819 does not teach which other of the over 900 sequences it provides would be lethal in corn rootworm species when used as dsRNA or siRNA, or even that they are useful in any other way. US Patent Application Publication No. U.S. 2013/040173 and PCT Application Publication No. WO 2013/169923 describe the use of sequences derived from the Snf7 gene of Diabrotica virgifera for RNA interference in maize. (Also published in Bolognesi et al., (2012) PLoS ONE7(10):e47534.doi:10.1371/journal.pone.0047534).

The vast majority of sequences complementary to corn rootworm DNA, such as the foregoing, when used as dsRNA or siRNA, do not provide protection for plants from corn rootworm species. For example, Baum et al., (2007) Nature Biotechnology 25: 1322-1326 describe the effect of RNAi inhibition of several WCR gene targets. These authors report that at extremely high iRNA (eg, dsRNA) concentrations exceeding 520 ng/ ^cm2 , 8 of the 26 target genes they tested failed to provide experimentally significant coleopteran pest mortality.

The authors of US Patent No. 7,612,194 and US Patent Publication No. 2007/0050860 were the first to report phytogenetic RNAi targeting western corn rootworm in corn plants. Baum et al., (2007) Nat. Biotechnol. 25(11):1322-6. These authors describe a high-throughput in vivo dietary RNAi system used to screen potential target genes for the development of transgenic RNAi maize. Of the initial gene pool of 290 targets, only 14 showed potential to control larvae. One of the most potent double-stranded RNAs (dsRNAs) targets the gene encoding vacuolar ATPase subunit A (V-ATPase), causing rapid repression of the corresponding endogenous mRNA at low concentrations of dsRNA and triggering specificity RNAi response. Thus, these authors document for the first time the potential of RNAi in the plant kingdom as a viable pest management tool, while at the same time demonstrating that effective targets cannot be accurately identified a priori even from relatively small candidate genomes.

Contents of the invention

Disclosed herein are nucleic acid molecules (e.g., target genes, DNA, dsRNA, siRNA, miRNA, shRNA, and hpRNA) and methods for their use in controlling insect pests, including, for example, the corn rootworm (Western corn rootworm, "WCR"), Butterfly beetle (Northern corn rootworm, "NCR"), Eleven-star beetle (Southern corn rootworm, "SCR"), Mexican corn rootworm (Mexican rootworm Insects, "MCR") Cucumber root beetles, Cucumber 11-star beetles, D.u. undecimpunctata Mannerheim, and South American beetles. In particular examples, exemplary nucleic acid molecules are disclosed that are homologous to at least a portion of one or more nucleic acids native to insect pests.

In these and other examples, the native nucleic acid sequence can be a target gene, the product of which can be involved, for example, but not limited to, in a metabolic process, or in larval development. In some instances, post-transcriptional inhibition of expression of a target gene by a nucleic acid molecule comprising a polynucleotide homologous to the target gene may be fatal to the insect pest, or may result in reduced growth and/or reduced viability of the insect pest. In a specific example, a transcription elongation factor, referred to herein as eg spt6 or an spt6 homologue, may be selected as a target gene for post-transcriptional silencing. In a specific example, a target gene useful for post-transcriptional repression is the gene referred to herein as zebra beetle spt6-1 (eg, SEQ ID NO: 1). Thus disclosed herein are isolated nucleic acid molecules comprising a polynucleotide of SEQ ID NO: 1 , the complement of SEQ ID NO: 1 and/or a fragment of any of the foregoing (eg, SEQ ID NO: 3-5).

Also disclosed are nucleic acid molecules comprising a polynucleotide encoding a polypeptide that is at least about 85% identical to an amino acid sequence within a target gene product (eg, the product of the spt6 gene). For example, a nucleic acid molecule may comprise a polynucleotide encoding a polypeptide at least 85% identical to SEQ ID NO: 2 (Pyrophyllum spt6-1), and/or a polypeptide within the product of Chromophyllia spt6-1 amino acid sequence. Further disclosed are nucleic acid molecules comprising a polynucleotide that is the reverse complement of a polynucleotide encoding a polypeptide that is at least 85% identical to an amino acid sequence within a target gene product.

Also disclosed are cDNA polynucleotides useful for generating iRNA (eg, dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecules that are complementary to all or part of an insect pest target gene (eg, the spt6 gene). In particular embodiments, dsRNA, siRNA, shRNA, miRNA and/or hpRNA can be produced in vitro or in vivo by genetically modifying organisms such as plants or bacteria. In a specific example, cDNA molecules are disclosed that can be used to generate iRNA molecules that are complementary to all or a portion of the spt6 gene (eg, SEQ ID NO: 1).

Further disclosed are spt6 components for repressing expression of essential genes in coleopteran pests, and spt6 components for providing protection to plants from coleopteran pests. An spt6 building block for inhibiting expression of essential genes in coleopteran pests is a single- or double-stranded RNA molecule consisting of a polynucleotide selected from the group consisting of SEQ ID NOs: 81-84 and complementary sequences thereof. Functional equivalents of spt6 components useful for suppressing expression of essential genes in coleopteran pests include all combinations with RNA transcribed from a coleopteran spt6 gene comprising SEQ ID NO:3, SEQ ID NO:4 and/or SEQ ID NO:5 Or part of a substantially homologous single- or double-stranded RNA molecule. An spt6 component for providing protection to plants from coleopteran pests is a DNA molecule comprising a polynucleoside operably linked to a promoter and encoding an spt6 component for inhibiting expression of an essential gene in a coleopteran pest acid, where the DNA molecule is able to integrate into the genome of the plant.

Disclosed are methods for controlling insect pests (e.g., Coleopteran pests) populations comprising providing an iRNA (e.g., dsRNA, siRNA, shRNA, miRNA, and hpRNA) molecule to an insect pest (e.g., Coleopteran pests), the iRNA molecule being activated by said After being ingested by the pest, it functions to inhibit biological functions in the pest, wherein the iRNA molecule comprises all or part of a polynucleotide selected from the group consisting of: SEQ ID NO:81; complementary sequence of SEQ ID NO:81; SEQ ID NO Complementary sequence of SEQ ID NO:82; SEQ ID NO:83; Complementary sequence of SEQ ID NO:83; SEQ ID NO:84; Complementary sequence of SEQ ID NO:84; Native spt6 with pest (such as WCR) A polynucleotide that hybridizes to a polynucleotide; a complementary sequence of a polynucleotide that hybridizes to a native spt6 polynucleotide of a pest; a polynucleotide that hybridizes to a polynucleotide naturally encoded by an organism of the genus Chrysophyll (such as WCR), which naturally The encoding polynucleotide comprises all or part of any one of SEQ ID NO: 1 and 3-5; the complement of a polynucleotide that hybridizes to a native encoding polynucleotide of an organism of the genus Chrysophyll, the native encoding polynucleotide Comprising all or part of any one of SEQ ID NO: 1 and 3-5.

In a particular embodiment, an iRNA that acts upon ingestion by an insect pest to inhibit a biological function in the pest is transcribed from a DNA comprising all or part of a polynucleotide selected from the group consisting of SEQ ID NO: 1 ; complementary sequence of SEQ ID NO: 1; SEQ ID NO: 3; complementary sequence of SEQ ID NO: 3; SEQ ID NO: 4; complementary sequence of SEQ ID NO: 4; SEQ ID NO: 5; SEQ ID NO: 5 Complementary sequence of Chromophyll organisms (such as WCR) naturally encoded polynucleotides comprising all or part of any one of SEQ ID NO: 1 and 3-5; Native encoded polynucleotides of Chromophyll organisms The complementary sequence of an acid, the naturally-encoded polynucleotide comprising all or part of any one of SEQ ID NO: 1 and 3-5.

Also disclosed herein are methods wherein the dsRNA, siRNA, shRNA, miRNA and/or hpRNA. In these and additional examples, the dsRNA, siRNA, shRNA, miRNA and/or hpRNA are ingestible by the pest. Ingestion of the dsRNA, siRNA, shRNA, miRNA and/or hpRNA of the invention can then cause RNAi in the pest, which in turn can result in the silencing of genes essential for the viability of the pest, ultimately causing the death of the pest. Accordingly, methods are disclosed wherein an insect pest is provided with a nucleic acid molecule comprising exemplary polynucleotides useful for controlling the insect pest. In specific examples, the coleopteran pests controlled by using the nucleic acid molecules of the present invention may be WCR, NCR, SCR, 11-star root beetle, cucumber 11-star beetle, cucumber 11-star beetle, South American Leaf beetles and D.u. undecimpunctata.

The foregoing and other features will become more apparent with reference to the following detailed description of several embodiments taken in conjunction with the accompanying drawings 1-2.

Description of drawings

Figure 1 includes a description of the strategy used to deliver dsRNA from a single transcription template with a single pair of primers.

Figure 2 includes a description of the strategy used to provide dsRNA from two transcriptional templates.

sequence listing

Nucleic acid sequences listed in the accompanying Sequence Listing are shown using standard letter abbreviations for nucleotide bases as specified in 37 C.F.R. §1.822. The listed nucleic acid and amino acid sequences define molecules (ie, polynucleotides and polypeptides, respectively) having nucleotide monomers and amino acid monomers arranged in the manner described. The listed nucleic acid sequences and amino acid sequences also each define a class of polynucleotides or polypeptides comprising nucleotide monomers and amino acid monomers arranged in the described manner. Taking into account the redundancy of the genetic code, it is understood that a nucleotide sequence comprising a coding sequence also describes a polynucleotide which encodes the same polypeptide as the polynucleotide of which the reference sequence consists. It is also understood that an amino acid sequence describes such a polynucleotide ORF that encodes the polypeptide.

Only one strand of each nucleic acid sequence is shown and any reference to the strand shown should be understood to include the complementary strand. Since the complement and reverse complement of a primary nucleic acid sequence are necessarily disclosed by that primary sequence, any reference to a nucleic acid sequence also includes the complement and reverse complement of that nucleic acid sequence, unless expressly stated otherwise (Or it's clear from the context in which the sequence occurs that it isn't). Furthermore, as understood in the art, the nucleotide sequence of an RNA strand is determined by the sequence of the DNA transcribed into that RNA strand (but substituting the uracil (U) nucleobase for thymine (T)), so any pair encoding References to DNA sequences of RNA sequences include such RNA sequences. In the accompanying sequence listing:

SEQ ID NO: 1 shows a contig containing exemplary WCR spt6 DNA (referred to as WCRspt6 or WCR spt6-1 in some places herein):

GTCACTTGTCAATTGTCAACCTGGAAAACGACTTGTCGAAGTCGCATAGTTTTTATAAGTTTAAATAAACTAAATTAAATATAAATACTTCGAGAATGCAATAATTATTATTCTTTAACTAGACCCACAGCTTATTAATTAGCAGAAGTAGTAGCAGACTTATACTAACTAGCATAAGGAGAAACATATTAACATAACATGGCAGACTTCATAGATTCTGAAGCAGAAGAAAGTAGTGAGGAGGAGGAATTAGATCATAGGGATCGTAAAAAAGCCCAAAAAGCCAAAGTTGTAGATAGTTCAGATGAAGATGATGAAGATGATGACGAAAGACTGAGAGAGGAATTAAAGGATTTGATTGATGATAATCCTATTGAAGAAAGTGATGCTGAGTCTGATGCTTCAGGAAGGGAAAAACGTAAGAAATCTGACGACGAGGATTTGGATGATCGACTGGAAGATGAAGATTATGATTTGCTTGAAGAAAATTTGGGTGTTAAAGTTGAAAGAAGGAAATTCAAGCGACTGCGGCGTTTTGAAGATGAAGAAAGTGAAGGAGAAGAAGAACATGATCCTGAACAAGATAGGGAACAAATTGCTATGGATATATTTTCAGATGATGACGATGAAAGACGATCAGAACGAAGTCACAGACCTGCCGTCGAACAAGAAACTTATGGTGTAGGCGAGGAAGAAGAGGAAGGGGAGTACTCGGATGCCGATGATTTTATAGTTGACGATGACGGTAGACCGATAGCTGAAAAGAAGAAGAAGAAAAAACCAATATTTACTGATGCCGCTCTCCAAGAGGCTCAAGAAACCTTCGGTGTCGATTTTGATTATGATGAATTTAGTAAATACGATGAAGATGATTACGAAGATGAAGAGGAGGAGGATGACGAATACGAGGAAGATGATGTAGAGAAAAGGAAACGGCCTAAAAAGACTTCAAAGAAAAAACCGACGAAGAAATCCATTTTTGAAGTGTATGAACCTAGTG AACTTAAAAGAGGGTTCTTTACCGATCTCGATAATGAAATCCGAAACACTGATATTCCCGAAAGAATGCAACTTCGTGATGTTCCAATCACCGCTGTTCCGGATGACTCAACTGAACTTGATGATGAAGCAGAATGGATTTACAGGCAAGCGTTTTGTAACAGAACTGTTTCCAATGTGGATTCTCATTTATCATCAGAGGCAAGAGAGAAATTAAAGAAGACTCATCATGCCATCGGAAAAATCAGAAAAGCATTAGATTTTATAAGAAATCAACAATTAGAAGTACCGTTTATTGCTTTTTATAGAAAGGAGTATGTTCAACCGGAACTTAATATTAACGATTTGTGGAAAGTATATAAATACGATGCAAAGTGGTGCCAATTGAAAACACGCAAAGAAAACCTCTTGAAGCTTTTTGAAAAAATGAGGTCATATCAAACTGACCACATAATGAAAGATCCAGATGCACCAATTCCAGACAACCTTCGTATTATGACTGAGTCCGACATTGAGCGATTGAAAAATGTTCAAACCGCCGAAGAGTTAAATGACGTTCACAATCATTTCATTTTATATTATGCTGCAGATTTGCCAGCCATGCATGCCGCGTGGAGAGTCAAAGAAAGAGAAAGGAGAAGACAGGAAAGAAAGGAGGCTAGACTTAAGCTCATCGCTGAAAGTGAAGAAGGTGCTGAAATTCCTGAAGAGCCTGAGGAAATTGACGATGATGAACCAGAAGCTGAAACCTTAAAATACGCCAATAGATCAGGCAGCTATGCACTATGTAATAAAGGAGGTTTGGGTCCTCTAGCGAAGAAATTTGGTTTAACGCCTGAAGAATTTGCCGAAAACCTGAGAGATAATTATCAGAGGCACGAAGTAGATCAAGAACATATAGAACCTGTAGAAGTCGCTAAAGAATTCGTATCGCCGAAATTTCCTACAGTGGAAGAAGTGTTGCAAGCTACTAAACACATGGTAGCTTTACAAATAGCAAG AGAACCATTAGTAAGGAAATGCGTAAGAGAAATTTTCTTTGAACGAGCTAGATTAAACGTTTATCCAACCAAAAAGGGTGTGAAAGTTATAGATGAAGCTCATAATTGCTATAGTATGAAGTATGTAAAAAATAAACCAGTAAGAGATCTTGCAGGCGACCAATTTTTAAAATTATGTTTAGCCGAAGAGGAGAACCTCCTTACTATTACCATCAATGACCATATTCAAGGAAACACTACTAACAATTACATTGATGAAGTCAAACAATTATATATAAAGGATGAATTCAGCAAACACGTTCAGGATTGGAATGCGCTAAGAATGGAATCGGTAGAAAGGGCTTTAACGAAAAGTGTCTTACCAGATTTAAGATCTGAATTAAAACGAACGTTGCTCACAGAGGCTAAAGAATTCGTATTAAAAGCTTGCTGTAGAAAATTATATAATTGGATAAAGATTGCTCCATACGCAATAACTTTTCCCGATGAAGACGAAGATGATTGGGATACATCCAAAGGTGTTAGAACTATGGGTGTAGCATACGTACCAGAGCACACAGTATCAGCTTTTACCTGTATTTCAGCACCAGACGGAGATATAACTGATTATCTCAGATTACCAAATATTCTAAAAAGAAAAAATAGCTTTCGAACTGAAGAAAAACTTATGAAGGAAGCTGATCTTCAAGCACTGAAAAATTTCATATTCCTTAAAAAGCCTCATGTGATAGCAGTAGGGGGTGAGTCCAGAGAAGCCTTAATGATCGCTGATGATATCAGAGGAGTAATAAGTGAACTAATAGAATCCGACCAATTCCCACAGATTAGGGTTGAAATTATTGATAATGAATTAGCCAAAGTGTACGCAAATTCCATTAAAGGTTCAACTGATTTCAGAGATTATCCAGAGTTGTTAAGGCAAGCTATTTCATTAGCTCGAAGAATGCAAGATCCTTTGGTTGAATTTTCCCAATTATGTAATAGCGATGAGGAAATTTTG AGTCTGAGGTTTCATCCCTTGCAGGAACAAGTCCAGAAAGAAGAACTACTAGAAGCTCTCTGTTTAGAATTTGTCAACAGAACAAATGAAGTAGGTGTAGATATAAATCTTGCCGTTCAGCAGATTCATAAAAGTAGTTTAGTTCAATTCATATGCGGTCTAGGACCGCGTAAAGGTCAAGCGTTACTTAAAGTTCTGAAACAAACTAATCAGAGGCTTGAAAACAGAACCCAATTGGTTACATTTTGTCATATGGGTCCAAAAGTTTTTATTAATTGTTCTGGATTCATAAAGATTGATACCAATAGTTTAGGAGACAGTACTGAGGCATATGTTGAAATATTGGATGGCTCTCGAGTTCATCCCGAAACTTATGAATGGGCACGAAAAATGGCTGTGGATGCTTTAGAATATGACGATGATGAGGGAGCTAATCCGGCGGGAGCTTTAGAGGAAATTCTCGAGGCGCCAGAGAGGTTAAAAGATCTTGACTTGGATGCATTTGCGGAGGAATTGGAAAGACAAGGATTTGGTAACAAGAGTATAACATTGTATGACATTAGAGCAGAACTGAACTCGCGATATAAAGATTTGAGACAACCTTTTCGTTCTGCAAATCCTGAAGAACTATTCGATATGCTTACTAAAGAAACTCCCGAAACATTTTATATTGGAAAAATGGTTACATCTACCGTGTTTGGCATTGCAAGGAGAAAACCAAAGTCAGACCAGCTCGATCAAGCTAATCCGGTCCGTAATGACGAAACTGGTTTGTGGCAGTGCCCCTTCTGTTTGAAAAATGATTTTCCTGAATTATCTGATGTATGGAATCATTTTGATGCAGGAGCATGTCCTGGTCAAGCTACTGGAGTTAAACTAAGACTGGATAATGGTATATTAGGTTATATTTATATAAAAAATATAAGCGACAAACCAGTTGCTAATCCGGAAGAAAGAGTAGGCATAGGACAATTAATTCACTGTAGAATAATAAAAATTG ACGTAGAGAAGTTTAGTGTTGATTGTACATCGAAATCTAGCGATCTTGCTGATAAAAATCATGAATGGAGACCTCAACGAGATCCTCATTATGATCAAGAACGTGAAGACAAAGACAATCGATTAGAAGCCGAGAAGAAGAAAGAGAAACAGCGTATGACGTACATCAAGAGGGTTATCGTTCATCCGGCGTTCCATAACATATCCTATGCCGAAGCCGAAAAATGTATGGTTAATATGGACCAGGGTGAAGTTATCATTAGGCCTTCGAGTAAGGGTGCGGATCATTTGACCATTACTTGGAAGGTTACGGATGGAATCTATCAACACATTGATATTAAGGAACAAGGGAAAGTTAACGCTTTTTCTTTAGGAAAATCATTATGGATTGGAAATGAAGAATTTGAAGATCTTGATGAAATAATAGCTAGGCACGTTACACCAATGGCGGCGCATGCTAGAGATTTACTGTATTTTAGGTATTACAAAGATTTCCAAGGTGGTCATAAAGATAAGGCTGAGGAATATCTAAAAGATGAAAAAAAGAAAAATGCTTCTAAAATTCATTATGTAGTTAGTGCTGCTAAGAATATACCTGGTAAATTTCTTCTTTCGTACCTTCCACGCAACAAAGTTAGACACGAATATGTAACAGTTACTCCAGAAGGCTTTCGGTTTAGACAACAAATGTTTGATTCCGTTTCTTCGTTATTTAAATGGTTCAAAGAACATTTCCGGGAGCCTCCACCAGGCGGTGCAACACCAGGTAGCACGCCAAGGATGGCTTCAAGCAGAACTGGATATGGAAGTGCCACACCCGCGTATAGTATGAATAATGAAGCTATTCAAAGAGTTGCTCAAAATTTACCAAGTCATGTAGTTCAAGCGTTATCTGCTGCCACCAACCAAACTCCACATTATCCCCACACCCCTGGTTATGGAGGCAATTATATTAATACGCCTTACACTCCAAGTGGTCAAACTCCATACATGACTCCGTA CGCTACACCCCATACGCAGCAAACTCCCCGCTATGGTCATCAAACACCTTCCCAACACATGGCGAGCTCAGCACCACAAGGTCTCAACAATCCCTTTTTACATCCTGGCGCGGTGACTCCCTCCCAACGAACTCCTATTTATCGCAACCATCCTGCACAATCTCCAGTAATGCTTCCTACAAGCCCTGTACCAAGTCCAGGTTCCCAGAGTTCATACAGTAGTCATTTAAGTCATAATCAGCGAAGTGGAAGTTATGCTGAATCCTTAAGATTCCAACCTCCCGAATCGCCGAGAAGCTCAGTGAGTAATAGAAGCTTTCAAACTGATAGATACGGCGGTGATAGGTATGGTAAAGGAGGAAGTCATAGATATGGTGGAAGCTCAAACGAAGATAGATATGGTAAGGGAGGAGGAGGAAATGAAAATACGGATTGGCAGAAAGCTGCAGAAGCATGGGCTCGATCTAGATCTACTCCGAGAAGTGATGGCCGTAATACTCCAAGATCTGTAGGTCAACGAACTCCTAGATACGACAACGACGCCGAACGTTCTAGAATGAAACACCTCAGCAAGAGCCCGAGGTCTGTTAGGTCTACTCCTCGAACCAATACATCTCCACATTCTATGTCCCTAGGTGATGCTACCCCTCTGTATGACGAAAGTATCTAAATTACTTTGTATTTGGTTATATAGTAGGGTAACAGTGATATTGTAAATGCTAAAATCAACAATACAGATTCGCGATATTAAGTCACAATGACGACCTCTCGTGGAGTTCGGTGAAACTAACTTTGAGGGCGTTTTCACTGTCAAACGTCCAGCGCACATGCACCTGAAACAAGTAGGCAATATTCATTATTTATTTTTACAGGTTTTTTATACAAATATTATTTACAACATAGACTTCAACTTTTACTGAAAGGTGAAGCTTTCATATTAATAATTTT

SEQ ID NO: 2 sets forth the amino acid sequence of the SPT6 polypeptide encoded by an exemplary WCR spt6 DNA (referred to as WCRSPT6 or WCR SPT6-1 in some places herein):

MADFIDSEAEESSEEEELDHRDRKKAQKAKVVDSSDEDDEDDDERLREELKDLIDDNPIEESDAESDASGREKRKKSDDEDLDDRLEDEDYDLLEENLGVKVERRKFKRLRRFEDEESEGEEEHDPEQDREQIAMDIFSDDDDERRSERSHRPAVEQETYGVGEEEEEGEYSDADDFIVDDDGRPIAEKKKKKKPIFTDAALQEAQETFGVDFDYDEFSKYDEDDYEDEEEEDDEYEEDDVEKRKRPKKTSKKKPTKKSIFEVYEPSELKRGFFTDLDNEIRNTDIPERMQLRDVPITAVPDDSTELDDEAEWIYRQAFCNRTVSNVDSHLSSEAREKLKKTHHAIGKIRKALDFIRNQQLEVPFIAFYRKEYVQPELNINDLWKVYKYDAKWCQLKTRKENLLKLFEKMRSYQTDHIMKDPDAPIPDNLRIMTESDIERLKNVQTAEELNDVHNHFILYYAADLPAMHAAWRVKERERRRQERKEARLKLIAESEEGAEIPEEPEEIDDDEPEAETLKYANRSGSYALCNKGGLGPLAKKFGLTPEEFAENLRDNYQRHEVDQEHIEPVEVAKEFVSPKFPTVEEVLQATKHMVALQIAREPLVRKCVREIFFERARLNVYPTKKGVKVIDEAHNCYSMKYVKNKPVRDLAGDQFLKLCLAEEENLLTITINDHIQGNTTNNYIDEVKQLYIKDEFSKHVQDWNALRMESVERALTKSVLPDLRSELKRTLLTEAKEFVLKACCRKLYNWIKIAPYAITFPDEDEDDWDTSKGVRTMGVAYVPEHTVSAFTCISAPDGDITDYLRLPNILKRKNSFRTEEKLMKEADLQALKNFIFLKKPHVIAVGGESREALMIADDIRGVISELIESDQFPQIRVEIIDNELAKVYANSIKGSTDFRDYPELLRQAISLARRMQDPLVEFSQLCNSDEEILSLRFHPLQEQVQKEELLEALCLEFVNRTNEVGVDINLAVQQIHKSSLVQFICGLGPRKGQALLKVL KQTNQRLENRTQLVTFCHMGPKVFINCSGFIKIDTNSLGDSTEAYVEILDGSRVHPETYEWARKMAVDALEYDDDEGANPAGALEEILEAPERLKDLDLDAFAEELERQGFGNKSITLYDIRAELNSRYKDLRQPFRSANPEELFDMLTKETPETFYIGKMVTSTVFGIARRKPKSDQLDQANPVRNDETGLWQCPFCLKNDFPELSDVWNHFDAGACPGQATGVKLRLDNGILGYIYIKNISDKPVANPEERVGIGQLIHCRIIKIDVEKFSVDCTSKSSDLADKNHEWRPQRDPHYDQEREDKDNRLEAEKKKEKQRMTYIKRVIVHPAFHNISYAEAEKCMVNMDQGEVIIRPSSKGADHLTITWKVTDGIYQHIDIKEQGKVNAFSLGKSLWIGNEEFEDLDEIIARHVTPMAAHARDLLYFRYYKDFQGGHKDKAEEYLKDEKKKNASKIHYVVSAAKNIPGKFLLSYLPRNKVRHEYVTVTPEGFRFRQQMFDSVSSLFKWFKEHFREPPPGGATPGSTPRMASSRTGYGSATPAYSMNNEAIQRVAQNLPSHVVQALSAATNQTPHYPHTPGYGGNYINTPYTPSGQTPYMTPYATPHTQQTPRYGHQTPSQHMASSAPQGLNNPFLHPGAVTPSQRTPIYRNHPAQSPVMLPTSPVPSPGSQSSYSSHLSHNQRSGSYAESLRFQPPESPRSSVSNRSFQTDRYGGDRYGKGGSHRYGGSSNEDRYGKGGGGNENTDWQKAAEAWARSRSTPRSDGRNTPRSVGQRTPRYDNDAERSRMKHLSKSPRSVRSTPRTNTSPHSMSLGDATPLYDESI

SEQ ID NO: 3 shows an exemplary WCR spt6 DNA used in some instances to produce dsRNA, referred to in several places herein as WCR spt6-1 reg1 (region 1):

CGCCTTACACTCCAAGTGGTCAAACTCCATACATGACTCCGTACGCTACACCCCATACGCAGCAAACTCCCCGCTATGGTCATCAAACACCTTCCCAACACATGGCGAGCTCAGCACCACAAGGTCTCAACAATCCCTTTTTACATCCTGGCGCGGTGACTCCCTCCCAACGAACTCCTATTTATCGCAACCATCCTGCACAATCTCCAGTAATGCTTCCTACAAGCCCTGTACCAAGTCCAGGTTCCCAGAGTTCATACAGTAGTCATTTAAGTCATAATCAGCGAAGTGGAAGTTATGCTGAATCCTTAAGATTCCAACCTCCCGAATCGCCGAGAAGCTCAGTGAGTAATAGAAGCTTTCAAACTGATAGATACGGCGGTGATAGGTATGGTAAAGGAGGAAGTCATAGATATGGTGGAAGCTCAAACGAAGATAGATATGGTAAGGGAGGAGGAGGAAATGAAAATACGGATTGGCAGAAAGCTGCAGAAGCATG

SEQ ID NO: 4 shows an additional exemplary WCR spt6 DNA used in some instances to produce dsRNA, referred to in some places herein as WCR spt6-1 v1 (version 1):

GACTCCGTACGCTACACCCCATACGCAGCAAACTCCCCGCTATGGTCATCAAACACCTTCCCAACACATGGCGAGCTCACACCACAAGGTCTCAAACAATCCCTTTTTACATCCTGGCGCGGTGACTCCCTCCCAACGAACTCCT

SEQ ID NO:5 shows an additional exemplary WCR spt6 DNA used in some instances to produce dsRNA, referred to as WCR spt6-1 v2 (version 2) in some places herein:

AGAGTTCATACAGTAGTCATTTAAGTCATAATCAGCGAAGTGGAAGTTATGCTGAATCCTTAAGATTCCAACCTCCCGAATCGCCGAGAAGCTCAGTGAGTAATAGAA

SEQ ID NO: 6 shows the nucleotide sequence of the T7 phage promoter.

SEQ ID NO: 7 shows a fragment of an exemplary YFP coding region.

SEQ ID NOs: 8-13 show primers used to amplify portions of exemplary WCR spt6 polynucleotides used in some examples to generate dsRNA, including spt6-1 reg1, spt6-1 v1 and spt6-1 v2.

SEQ ID NO: 14 shows an exemplary YFP gene.

SEQ ID NO: 15 shows the DNA sequence of Annexin Region 1.

SEQ ID NO: 16 shows the DNA sequence of annexin region 2.

SEQ ID NO: 17 shows the DNA sequence of region 1 of β-spectrin 2.

SEQ ID NO: 18 shows the DNA sequence of β-spectrin 2 region 2.

SEQ ID NO: 19 shows the DNA sequence of region 1 of mtRP-L4.

SEQ ID NO: 20 shows the DNA sequence of mtRP-L4 region 2.

SEQ ID NOs: 21-48 show primers used to amplify the gene regions of Annexin, β-spectrin 2, mtRP-L4 and YFP to synthesize dsRNA.

SEQ ID NO: 49 shows the maize DNA sequence encoding a TIP41-like protein.

SEQ ID NO:50 shows the nucleotide sequence of the T20VN primer oligonucleotide.

SEQ ID NOs: 51-61 show primers and probes for analysis of dsRNA transcript expression in maize.

SEQ ID NO: 62 shows the nucleotide sequence of a portion of the SpecR coding region used to detect the backbone of the binary vector.

SEQ ID NO: 63 shows the nucleotide sequence of the AAD1 coding region used to analyze the genome copy number.

SEQ ID NO:64 shows the DNA sequence of the maize invertase gene.

SEQ ID NOs: 65-73 show the nucleotide sequences of DNA oligonucleotides used to determine gene copy number and detect binary vector backbones.

SEQ ID NOs: 74-79 show primers and probes that can be used in some embodiments for the analysis of dsRNA transcript maize expression.

SEQ ID NO:80 shows an exemplary linker polynucleotide that forms a "loop" when transcribed in an RNA transcript to form a hairpin structure:

AGTCATCACGCTGGAGCGCACATAGGCCCTCCATCAGAAAGTCATTGTGTATATCTCTCATAGGGAACGAGCTGCTTGCGTATTTCCCTTCCGTAGTCAGAGTCATCAATCAGCTGCACCGTGTCGTAAAGCGGGACGTTCGCAAGCTCGT

SEQ ID NOs:81-84 show RNA transcribed from nucleic acids comprising exemplary spt6 polynucleotides and fragments thereof.

Detailed ways

I. Overview of several implementations

We developed RNA interference (RNAi) as a tool for managing insect pests using dsRNA-expressing transgenic plants, one of the most likely targeted pest species, the western corn rootworm. To date, most of the genes proposed as targets for RNAi in rootworm larvae have not actually fulfilled their purpose. We describe herein RNAi-mediated knockdown of a transcription elongation factor (spt6) in the exemplary insect pest western corn rootworm, which knockdown occurs when, for example, an iRNA molecule is delivered via ingested spt6 dsRNA. Shown to have a lethal phenotype. In embodiments herein, the ability to deliver spt6 dsRNA to insects via feeding confers RNAi effects that are very useful for managing insect (eg, Coleopteran) pests. By combining spt6-mediated RNAi with other useful RNAi targets (e.g., ROP RNAi targets, as described in U.S. Patent Application No. 14/577,811; RNA polymerase I1 RNAi targets, as described in U.S. Patent Application No. 62/133,214; RNA polymerase II140 RNAi target as described in U.S. Patent Application No. 14/577,854; RNA Polymerase II215 RNAi target as described in U.S. Patent Application No. 62/133,202; RNA Polymerase II33 RNAi target as described in U.S. Patent Application No. 62/ 133,210, ncmRNAi target as described in U.S. Patent Application No. 62/095487; Dre4 RNAi target as described in U.S. Patent Application No. 14/705,807; COPIα RNAi target as described in U.S. Patent Application No. 62/063,199 COPIβRNAi target, as described in U.S. Patent Application No. 62/063,203; COPIγRNAi target, as described in U.S. Patent Application No. 62/063,192; COPIδ RNAi target, as described in U.S. Patent Application No. 62/063,216; and transcription elongation factor spt5 RNAi target, as described in U.S. Patent Application No. 62/168613), the potential to affect multiple target sequences in rootworms (e.g., larval state rootworms) could increase the development of sustainable insect pests involving RNAi technology Opportunities for management methods.

Disclosed herein are methods and compositions for the genetic control of insect (eg, Coleopteran) pest infestation. Also provided are methods for identifying one or more genes essential to the life cycle of an insect pest for use as target genes for RNAi-mediated control of insect pest populations. DNA plasmid vectors encoding RNA molecules can be designed to suppress one or more target genes necessary for growth, survival and/or development. In some embodiments, the RNA molecule may be capable of forming a dsRNA molecule. In some embodiments, there is provided a method of post-transcriptionally repressing the expression of a target gene or inhibiting a target gene via a nucleic acid molecule complementary to the coding or non-coding sequence of the target gene in an insect pest. In these and additional embodiments, a pest may ingest one or more dsRNA, siRNA, shRNA, miRNA, and/or hpRNA molecules transcribed from all or a portion of a nucleic acid molecule that is complementary to a coding or non-coding sequence of a target gene , thus providing plant protection effect.

Accordingly, some embodiments involve sequence-specific inhibition of expression of target gene products using dsRNA, siRNA, shRNA, miRNA, and/or hpRNA that are complementary to the coding and/or non-coding sequences of one or more target genes to achieve At least partial control of insect (eg Coleopteran) pests. Disclosed is a set of isolated and purified nucleic acid molecules comprising, for example, polynucleotides as set forth in SEQ ID NO: 1, fragments of SEQ ID NO: 1, and complementary sequences of the foregoing. In some embodiments, stabilized dsRNA molecules can be expressed from these polynucleotides, fragments thereof, or genes comprising one of these polynucleotides for post-transcriptional silencing or inhibition of target genes. In certain embodiments, the isolated and purified nucleic acid molecule comprises all or a portion of any of SEQ ID NOs: 1 and 3-5 and/or complements thereof.

Some embodiments relate to recombinant host cells (eg, plant cells) having in their genome at least one recombinant DNA encoding at least one iRNA (eg, dsRNA) molecule. In particular embodiments, encoded dsRNA molecules may be provided upon ingestion by an insect (eg, Coleopteran) pest for post-transcriptional silencing or inhibition of expression of a target gene in the pest. The recombinant DNA may comprise, for example: any one of SEQ ID NO: 1 and 3-5; a fragment of any one of SEQ ID NO: 1 and 3-5; and a fragment comprising SEQ ID NO: 1 and 3- 5 and/or a polynucleotide consisting of a partial sequence of a gene of one of its complementary sequences.

Some embodiments relate to recombinant host cells having in their genome recombinant DNA encoding at least one iRNA (e.g., dsRNA) molecule comprising all or part of SEQ ID NO: 81 (e.g., selected from SEQ ID NO: 82 -84), or its complement. The one or more iRNA molecules can silence or inhibit target spt6 DNA (e.g., comprising all of the polynucleotides selected from SEQ ID NO: 1 and 3-5) when ingested by an insect (e.g., Coleopteran) pest. or part of the DNA) in the pest or progeny of the pest, thereby causing the growth, development, viability and/or feeding cessation of the pest.

In some embodiments, a recombinant host cell having in its genome at least one recombinant DNA encoding at least one RNA molecule capable of forming a dsRNA molecule can be a transformed plant cell. Some embodiments relate to transgenic plants comprising such transformed plant cells. In addition to such transgenic plants, progeny plants of any transgenic plant generation, transgenic seeds and transgenic plant products, each of which comprise one or more recombinant DNAs, are also provided. In specific embodiments, RNA molecules capable of forming dsRNA molecules can be expressed in transgenic plant cells. Thus, in these and other embodiments, dsRNA molecules can be isolated from transgenic plant cells. In a particular embodiment, the transgenic plant is a plant selected from Zea mays and Poaceae plants.

Some embodiments relate to methods for modulating the expression of a target gene in an insect (eg, Coleopteran) pest cell. In these and other embodiments, a nucleic acid molecule can be provided, wherein the nucleic acid molecule comprises a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule. In particular embodiments, a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule may be operably linked to a promoter, and may also be operably linked to a transcription termination sequence. In particular embodiments, the method for modulating the expression of a target gene in an insect pest cell may comprise: (a) transforming a plant cell with a vector comprising a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule; (b) in culturing the transformed plant cells under conditions sufficient to permit development of a plant cell culture comprising a plurality of transformed plant cells; (c) selecting for transformed plant cells that have integrated the vector into their genome; and (d) It is determined that the selected transformed plant cells comprise an RNA molecule capable of forming a dsRNA molecule encoded by the polynucleotide of the vector. Plants can be regenerated from plant cells having integrated the vector into the genome and comprising the dsRNA molecule encoded by the polynucleotide of the vector.

Thus, also disclosed is a transgenic plant having integrated in its genome a vector having a polynucleotide encoding an RNA molecule capable of forming a dsRNA molecule, wherein the transgenic plant comprises a dsRNA encoded by the polynucleotide of the vector molecular. In particular embodiments, expressing in a plant an RNA molecule capable of forming a dsRNA molecule is sufficient to regulate access to said transformed plant or plant cell (e.g., by using said transformed plant, a part of the plant (e.g., a root), or Expression of a target gene in the cells of an insect (eg, Coleoptera) pest that feeds on plant cells, such that the growth and/or survival of the pest is inhibited. The transgenic plants disclosed herein may exhibit protection and/or enhanced protection against infestation by insect pests. Certain transgenic plants may exhibit protection and/or enhanced protection against one or more Coleopteran pests selected from the group consisting of: WCR, NCR, SCR, MCR, Cucumber-root Beetle, Cucumber Eleven Star Leaf Beetle, South American Leaf Beetle and D.u. undecimpunctata Mannerheim.

Also disclosed herein are methods for delivering control agents, such as iRNA molecules, to insect (eg, Coleopteran) pests. Such control agents can directly or indirectly impair the ability of insect pest populations to feed, grow, or otherwise cause damage to the host. In some embodiments, a method is provided comprising delivering a stabilized dsRNA molecule to an insect pest to repress at least one target gene in the pest, thereby causing RNAi and reducing or eliminating plant damage in the pest host. In some embodiments, the method of inhibiting the expression of a target gene in an insect pest results in the arrest of growth, survival and/or development of the pest.

In some embodiments, compositions (e.g., topical compositions) comprising iRNA (e.g., dsRNA) molecules are provided for use in plants, animals, and/or in the environment of plants or animals for the purpose of eliminating or mitigating insects ( such as Coleoptera) pest infestation. In particular embodiments, the composition may be a nutritional composition or food source to be fed to an insect pest. Some embodiments include making the nutritional composition or food source available to the pest. Ingestion of a composition comprising an iRNA molecule can result in uptake of the molecule by one or more cells of the pest, which in turn can result in inhibition of expression of at least one target gene in the one or more cells of the pest. By providing one or more compositions comprising iRNA molecules in a host of the pest, damage to plants or plant cells due to insect pest infestation can be limited or eliminated in or on any host tissue or environment where the pest is present ingestion or damage.

RNAi decoys are formed when dsRNA is mixed with food or attractants or both. When the pest eats the bait, it also eats the dsRNA. Baits can take the form of granules, gels, flowable powders, liquids or solids. In particular embodiments, pt6 can be incorporated into bait formulations, such as described in US Pat. No. 8,530,440, which is hereby incorporated by reference. Generally, where baits are used, the baits are placed in or around the environment of the insect pest so that, for example, WCRs can access and/or be attracted to the baits.

The compositions and methods disclosed herein can be used in conjunction with other methods and compositions for controlling damage caused by insect (eg, Coleopteran) pests. For example, an iRNA molecule as described herein for protecting plants from insect pests can be used in a method comprising the additional use of one or more chemicals effective against insect pests, effective against such pests, biopesticides, crop rotations, exhibiting characteristics that differ from those of RNAi-mediated methods and RNAi compositions (e.g., recombinant gene technology in plants to recombinantly produce proteins (e.g., Bt toxins) that are harmful to insect pests, and/or recombinantly express other iRNA molecules.

II. Abbreviations

dsRNA double-stranded ribonucleic acid

GI growth inhibition

NCBI National Center for Biotechnology Information

gDNA genomic deoxyribonucleic acid

iRNA inhibitory ribonucleic acid

ORF open reading frame

RNAi ribonucleic acid interference

miRNA micro ribonucleic acid

shRNA small hairpin RNA

siRNA small inhibitory ribonucleic acid

hpRNA hairpin ribonucleic acid

UTR untranslated region

WCR Western Corn Rootworm (Corn Rootworm)

NCR Northern Corn Rootworm (Rootworm Butterfly)

MCR Mexican corn rootworm (Mexican corn rootworm)

PCR polymerase chain reaction

qPCR quantitative polymerase chain reaction

RISC RNA-induced silencing complex

SCR Southern Corn Rootworm (Eleven-star root firefly beetle)

SEM standard error of the mean

YFP yellow fluorescent protein

III. Terminology

In the descriptions and tables that follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given to such terms, the following definitions are provided:

Coleopteran pests: As used herein, the term "coleoptera pests" refers to pest insects of the order Coleoptera, including Chillibeetle pest insects that feed on agricultural crops and crop products, including corn and other true grasses. In a specific example, the coleopteran pest is selected from the following list: corn root beetle (WCR), barbet's root beetle (NCR), eleven star root beetle (SCR), Mexican corn root beetle (MCR), D. u. undecimpunctata Mannerheim, D. u. undecimpunctata Mannerheim.

Contact (with an organism): As used herein, terms such as "contact" with an organism (such as a coleopteran pest) or "ingested" by an organism, with respect to a nucleic acid molecule, include internalization of the nucleic acid molecule into the organism, e.g. But not limited to: uptake of the molecule by the organism (eg, by eating); contacting the organism with a composition comprising the nucleic acid molecule; and soaking the organism with a solution comprising the nucleic acid molecule.

Contig: As used herein, the term "contig" refers to a DNA sequence reconstructed from a set of overlapping DNA segments derived from a single genetic source.

Maize plant: As used herein, the term "maize plant" refers to a plant of the species Zea mays.

Expression: As used herein, "expression" of an encoding polynucleotide (e.g., gene or transgene) refers to the process by which the encoded information of a nucleic acid transcription unit (including, for example, gDNA or cDNA) is converted into an operative part of the cell , non-operating or structural parts, usually including protein synthesis. Gene expression can be affected by external signals, such as exposure of a cell, tissue or organism to agents that increase or decrease gene expression. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression, for example by controlling effects on transcription, translation, RNA transport and processing, degradation of intermediate molecules such as mRNA, or by activation, inactivation, compartmentalization or degradation of specific protein molecules after they have been produced, or by these combination occurs. Gene expression can be measured at the RNA level or at the protein level by any method known in the art, including but not limited to Northern blotting, RT-PCR, Western blotting, or in vitro, in situ, or in vivo protein activity assays.

Genetic material: As used herein, the term "genetic material" includes all genes and nucleic acid molecules, such as DNA and RNA.

Inhibition: As used herein, the term "inhibition" when used to describe an effect on an encoding polynucleotide (e.g., a gene) refers to the mRNA transcribed from the encoding polynucleotide and/or the peptide, polypeptide of the encoding polynucleotide Or the protein product is measurably reduced at the cellular level. In some instances, inhibiting expression of an encoding polynucleotide results in near disappearance of expression. By "specific inhibition" is meant inhibition of a target encoding polynucleotide in the cell in which specific inhibition is being achieved without consequent effect on the expression of other encoding polynucleotides (eg, genes).

Insects: As used herein, with reference to pests, the term "insect pests" specifically includes insect pests of the order Coleoptera. In some instances, the term "insect pest" refers specifically to pests of the genus Coleoptera selected from the following list: corn root beetle (WCR), barley root beetle (NCR), Chrysopora chinensis (SCR), Mexican corn-root beetle (MCR), Cucumber-root beetle, Cucumber 11-star beetle, South American beetle and D.u. undecimpunctata Mannerheim.

Isolated: An "isolated" biological component (such as a nucleic acid or protein) has been separated from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and protein ) are substantially isolated, produced separately, or purified while effecting a chemical or functional change in the components (eg, a nucleic acid can be isolated from a chromosome by breaking the chemical bonds that link the nucleic acid to the rest of the DNA in the chromosome). Nucleic acid molecules and proteins that have been "isolated" include nucleic acid molecules and proteins purified by standard purification methods. The term also includes nucleic acids and proteins produced by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules, proteins and peptides.

Nucleic acid molecule: As used herein, the term "nucleic acid molecule" may refer to a polymeric form of nucleotides, which may include both the sense and antisense strands of RNA, cDNA, gDNA, as well as synthetic forms and mixed polymers of the foregoing . Nucleotides or nucleobases may refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotides. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise indicated, nucleic acid molecules are generally at least 10 bases in length. By convention, the nucleotide sequence of a nucleic acid molecule is read from the 5' end to the 3' end of the molecule. The "complement" of a nucleic acid molecule refers to a polynucleotide having nucleobases that can form base pairs (ie, A-T/U and G-C) with the nucleobases of the nucleic acid molecule.

Some embodiments include a nucleic acid comprising template DNA transcribed into an RNA molecule that is the complement of an mRNA molecule. In these embodiments, the complementary sequence of the nucleic acid transcribed into the mRNA molecule is present in a 5' to 3' orientation such that RNA polymerase (which transcribes the DNA in the 5' to 3' direction) will transcribe a sequence from the complementary sequence that is compatible with the mRNA molecule. hybridized nucleic acids. Thus, unless expressly stated otherwise or clear from the context to indicate otherwise, the term "complementary sequence" refers to a sequence having, from 5' to 3', a nucleobase that can form a base pair with a nucleobase of a reference nucleic acid. polynucleotide. Similarly, unless expressly stated otherwise (or otherwise clear from the context), the "reverse complement" of a nucleic acid refers to the complementary sequence in the opposite orientation. The foregoing is demonstrated in the following diagram:

ATGATGATG polynucleotide

The "complement" of the TACTACTAC polynucleotide

The "reverse complement" of a CATCATCAT polynucleotide

Some embodiments of the invention may include RNAi molecules that form hairpin RNAs. In these RNAi molecules, both the complement of the nucleic acid targeted by the RNA interference and the reverse complement can be present in the same molecule, so that the single-stranded RNA molecule can be "folded" to include the complementary polynucleotide and the reverse complement. Hybridizes to and to itself on a region of a complementary polynucleotide.

"Nucleic acid molecule" includes all polynucleotides, for example: DNA in single- and double-stranded forms; RNA in single-stranded form; and RNA in double-stranded form (dsRNA). The term "nucleotide sequence" or "nucleic acid sequence" refers to both the sense and antisense strands of a nucleic acid as individual single strands or in a duplex. The term "ribonucleic acid" (RNA) includes iRNA (inhibitory RNA), dsRNA (double-stranded RNA), siRNA (small interfering RNA), shRNA (small hairpin RNA), mRNA (messenger RNA), miRNA (microRNA), hpRNA (hairpin RNA), tRNA (transfer RNA, either loaded or unloaded with the corresponding acylated amino acid) and cRNA (complementary RNA). The term "deoxyribonucleic acid" (DNA) includes cDNA, gDNA and DNA-RNA hybrids. The terms "polynucleotide" and "nucleic acid" and "fragments" thereof will be understood by those skilled in the art as terms that include both gDNA, ribosomal RNA, transfer RNA, messenger RNA, operons, and smaller An engineered polynucleotide (which encodes or can be adapted to encode a peptide, polypeptide or protein) of .

Oligonucleotide: Oligonucleotides are short nucleic acid polymers. Oligonucleotides can be formed by cleaving longer nucleic acid segments, or by polymerizing individual nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to several hundred bases in length. Since oligonucleotides can bind to complementary nucleic acids, they can be used as probes for detecting DNA or RNA. Oligonucleotides (oligodeoxyribonucleotides) composed of DNA are used in PCR, a technique for amplifying DNA. In PCR, oligonucleotides are often referred to as "primers," which allow DNA polymerases to extend the oligonucleotide and copy the complementary strand.

A nucleic acid molecule may comprise naturally occurring nucleotides and/or modified nucleotides linked together by naturally occurring nucleotide linkages and/or non-naturally occurring nucleotide linkages. As will be readily understood by those skilled in the art, nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with analogs, internucleotide modifications (e.g. uncharged linkages: e.g. methylphosphonate, triphosphate Esters, phosphoramidates, carbamates, etc.; charged linkages: such as phosphorothioate, phosphorodithioate, etc.; pendant parts: such as peptides; intercalating agents: such as acridine, psoralen, etc.; chelating agents ; alkylating agents; and modified linkages: eg α-anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin, circular and padlocked conformations.

As used herein, the terms "coding polynucleotide", "structural polynucleotide" or "structural nucleic acid molecule" with reference to DNA refer to a polynucleotide that, when placed under the control of appropriate regulatory elements, , through transcription and mRNA translation into polypeptides. With respect to RNA, the term "encoding polynucleotide" refers to a polynucleotide that is translated into a peptide, polypeptide or protein. The boundaries of the encoding polynucleotide are defined by a translation initiation codon at the 5'-terminus and a translation termination codon at the 3'-terminus. Encoding polynucleotides include, but are not limited to: gDNA, cDNA, EST, and recombinant polynucleotides.

As used herein, "transcribed non-coding polynucleotide" refers to segments of an mRNA molecule that are not translated into peptides, polypeptides or proteins, such as 5'UTRs, 3'UTRs, and intronic segments. Additionally, "transcribed non-coding polynucleotide" refers to a nucleic acid that is transcribed into RNA that functions in a cell, such as structural RNA (e.g. ribosomal RNA (rRNA), for example 5SrRNA, 5.8S rRNA, 16S rRNA, 18S rRNA, 23S rRNA, and 28S rRNA, etc.), transfer RNA (tRNA), and snRNA such as U4, U5, U6, etc. Transcribed non-coding polynucleotides also include, for example but not limited to, small RNA (sRNA), a term commonly used to describe small bacterial non-coding RNAs, small nucleolar RNAs (snoRNAs), microRNAs, small interfering RNAs (siRNAs) ), Piwi interacting RNAs (piRNAs), and long non-coding RNAs. Still further, "transcribed non-coding polynucleotide" refers to a polynucleotide that naturally occurs in a nucleic acid as a "spacer sequence" within a gene and is transcribed into an RNA molecule.

Lethal RNA interference: As used herein, the term "lethal RNA interference" refers to RNA interference that causes death or reduced viability of a subject individual to whom eg dsRNA, miRNA, siRNA, shRNA and/or hpRNA are delivered.

Genome: As used herein, the term "genome" refers to the chromosomal DNA present in the nucleus of a cell, and also refers to the organelle DNA present in the subcellular components of the cell. In some embodiments of the invention, a DNA molecule can be introduced into a plant cell such that the DNA molecule is integrated into the genome of the plant cell. In these and additional embodiments, the DNA molecule can be integrated into the nuclear DNA of the plant cell, or into the DNA of the chloroplasts or mitochondria of the plant cell. The term "genome" when applied to bacteria refers to both chromosomes and plasmids within the bacterial cell. In some embodiments of the invention, a DNA molecule can be introduced into a bacterium such that the DNA molecule is integrated into the genome of the bacterium. In these and additional embodiments, the DNA molecule can be integrated into the chromosome, or located as or within a stable plasmid.

Sequence identity: As used herein, the term "sequence identity" or "identity" in the context of two polynucleotides or polypeptides means the two polynucleotides or polypeptides when aligned for maximum correspondence over a specified comparison window. identical residues in the sequence of the molecule.

As used herein, the term "percent sequence identity" may refer to a value determined by comparing two optimally aligned sequences of molecules (eg, nucleic acid sequences or polypeptide sequences) over a comparison window, wherein in order to achieve optimal alignment of the two sequences Alignment, the portion of the sequence in the comparison window may contain additions or deletions (ie gaps) compared to a reference sequence (which does not contain additions or deletions). The number of matching positions is generated by determining the number of positions at which the same nucleotide or amino acid residue occurs in the two sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to generate sequence identity percentage of sex to calculate the percentage. A sequence that is identical at every position compared to the reference sequence is considered to be 100% identical to the reference sequence, and vice versa.

Methods of alignment of sequences for comparison are well known in the art. Various programs and alignment algorithms are described in, for example, Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman ( 1988) Proc.Natl.Acad.Sci.U.S.A.85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5:151-3; Corpet et al., (1988) Nucleic Acids Res. 16: 10881-90; Huang et al., (1992) Comp. Appl. Biosci. 8: 155-65; Pearson et al. (1994) Methods Mol. Biol. 24: 307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. Detailed considerations of sequence alignment methods and homology calculations can be found, eg, in Altschul et al. (1990) J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST ^™ ; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD) and is available on the Internet. available online for use in conjunction with several sequence analysis programs. A description of how to use this program to determine sequence identity is available on the Internet in the "Help" section of BLAST ^(TM) . For comparison of nucleic acid sequences, the "Blast 2 Sequences" function of the BLAST ^™ (Blastn) program using the default BLOSUM62 matrix set as default parameters can be used. Nucleic acids having even greater sequence similarity to the sequence of a reference polynucleotide will show an increased percent identity when assessed by this method.

Specifically hybridizable/specifically complementary: As used herein, the terms "specifically hybridizable" and "specifically complementary" are terms that indicate a sufficient degree of complementarity sufficient to allow for a reaction between a nucleic acid molecule and a target nucleic acid molecule. Stable and specific binding occurs. Hybridization between two nucleic acid molecules involves the formation of an antiparallel alignment between the nucleobases of the two nucleic acid molecules. These two molecules are then able to form hydrogen bonds with corresponding bases on opposite strands to form a duplex molecule, which, if sufficiently stable, can be detected using methods well known in the art. A polynucleotide is not necessarily 100% complementary to its target nucleic acid to which it is specifically hybridizable. However, the amount of complementarity that must be present for specific hybridization will vary with the hybridization conditions used.

Hybridization conditions that result in a particular degree of stringency will vary depending on the nature of the hybridization method chosen and the composition and length of the hybridizing nucleic acid. Generally speaking, the temperature of hybridization and the ionic strength of the hybridization buffer (especially Na ⁺ and/or Mg ⁺⁺ concentration) will determine the stringency of hybridization, but the number of washes also affects stringency. Calculations of hybridization conditions required to achieve a particular degree of stringency are known to those of ordinary skill in the art and are discussed, for example, in: Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual , 2nd Edition, pp. Volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, Chapters 9 and 11; and Hames and Higgins (eds.), Nucleic Acid Hybridization , IRL Press, Oxford, 1985. Further details and guidance on nucleic acid hybridization can be found, for example, in Tijssen, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes , Part I, Chapter 2, Elsevier, NY, 1993; and Ausubel et al. (eds), Current Protocols in Molecular Biology , Chapter 2, Greene Publishing and Wiley-Interscience, NY, 1995.

As used herein, "stringent conditions" encompass conditions under which hybridization occurs only when there is less than 20% mismatch between the sequence of the hybridizing molecule and a homologous polynucleotide within the target nucleic acid molecule. "Stringent conditions" include additional specific levels of stringency. Thus, as used herein, conditions of "medium stringency" are conditions under which molecules with more than 20% mismatches in sequences will not hybridize; conditions of "high stringency" are conditions under which sequences with more than 10% mismatches will not hybridize; High stringency"conditions are those under which sequences with more than 5% mismatch will not hybridize.

The following are representative non-limiting hybridization conditions.

High stringency conditions (detection of polynucleotides sharing at least 90% sequence identity): hybridization in 5x SSC buffer at 65°C for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 min; and two washes in 0.5x SSC buffer at 65°C for 20 min each.

Moderate stringency conditions (polynucleotides sharing at least 80% sequence identity are detected): hybridize in 5x-6x SSC buffer at 65-70°C for 16-20 hours; wash in 2x SSC buffer at room temperature Two washes of 5-20 min each; and two washes in 1x SSC buffer at 55-70°C for 30 min each.

Non-stringent control conditions (polynucleotides sharing at least 50% sequence identity will hybridize): hybridize in 6x SSC buffer at room temperature to 55 °C for 16-20 hours; in 2x-3x SSC buffer at room temperature to 55 °C Wash at least twice at ℃, 20-30 minutes each time.

As used herein, the terms "substantially homologous" or "substantial homology" with respect to nucleic acids refer to polynucleotides having contiguous nucleobases that hybridize under stringent conditions to a reference nucleic acid. For example, a nucleic acid substantially homologous to a reference nucleic acid represented by any one of SEQ ID NOs: 1 and 3-5 hybridizes to the reference nucleic acid under stringent conditions (e.g., the medium stringency conditions set forth above) of those nucleic acids. Substantially homologous polynucleotides may have at least 80% sequence identity. For example, substantially homologous polynucleotides may have about 80% to 100% sequence identity, such as 79%, 80%, about 81%, about 82%, about 83%, about 84%, about 85%, About 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98 %, about 98.5%, about 99%, about 99.5%, and about 100%. The property of substantial homology is closely related to specific hybridization. For example, a nucleic acid molecule can specifically hybridize when a sufficient degree of complementarity exists to avoid non-specific binding of the nucleic acid to a non-target polynucleotide where specific binding is desired (eg, under stringent hybridization conditions).

As used herein, the term "ortholog" refers to a gene in two or more species that has evolved from a common ancestral nucleic acid and that may retain the same function in the two or more species.

As used herein, two nucleotides are complementary when each nucleotide of a polynucleotide read in the 5' to 3' direction is complementary to each nucleotide of another polynucleotide when read in the 3' to 5' direction. nucleic acid molecules are said to exhibit "perfect complementarity". A polynucleotide that is complementary to a reference polynucleotide will exhibit the same sequence as the reverse complement of said reference polynucleotide. These terms and descriptions are clearly defined in the art and are easily understood by those of ordinary skill in the art.

Operably linked: A first polynucleotide is said to be operably linked to a second polynucleotide when it has a functional relationship with the second polynucleotide. When produced recombinantly, operably linked polynucleotides will generally be contiguous and, where necessary, join two protein coding regions in the same reading frame (eg, in a translationally fused ORF). However, operably linked nucleic acids do not have to be contiguous.

The term "operably linked" when used in relation to a regulatory genetic element and an encoding polynucleotide means that the regulatory element affects the expression of the linked encoding polynucleotide. "Regulatory element" or "control element" refers to a polynucleotide that affects the timing and level/amount of transcription, RNA processing or stability, or translation of an associated encoding polynucleotide. Regulatory elements may include promoters, translation leader sequences, introns, enhancers, stem-loop structures, repressor binding polynucleotides, polynucleotides with termination sequences, polynucleotides with polyadenylation recognition sequences, and many more. A particular regulatory element may be located upstream and/or downstream of the encoding polynucleotide to which it is operably linked. Furthermore, specific regulatory elements operably linked to the encoding polynucleotide may be located on the relevant complementary strand of the double-stranded nucleic acid molecule.

Promoter: As used herein, the term "promoter" refers to a region of DNA that may be upstream from the initiation of transcription and may be involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter may be operably linked to a polynucleotide encoding a signal peptide for expression in a cell, or a promoter may be operably linked to a polynucleotide encoding a signal peptide which may be operably linked to a polynucleotide for expression in a cell The encoding polynucleotides are operably linked. A "plant promoter" may be a promoter capable of initiating transcription in a plant cell. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as "tissue-preferred" promoters. Promoters that initiate transcription only in certain tissues are referred to as "tissue-specific" promoters. A "cell type specific" promoter primarily drives expression in certain cell types (eg, vascular cells in roots or leaves) in one or more organs. An "inducible" promoter can be one that can be placed under environmental control. Examples of environmental conditions under which transcription can be initiated by an inducible promoter include anaerobic conditions and the presence of light. Tissue-specific, tissue-preferred, cell-type-specific, and inducible promoters constitute the class of "non-constitutive" promoters. A "constitutive" promoter is one that is active under most environmental conditions or in most tissues or cell types.

Any inducible promoter may be used in some embodiments of the invention. See Ward et al., (1993) Plant Mol. Biol. 22:361-366. With an inducible promoter, the rate of transcription increases in response to the inducing agent. Exemplary inducible promoters include, but are not limited to: a copper-responsive promoter from the ACEI system; an In2 gene promoter from maize that is responsive to the benzenesulfonamide herbicide safener; a Tet repressor from Tn10; and a steroid-derived promoter. An inducible promoter of a hormone gene, the transcriptional activity of which is induced by glucocorticoid hormones (Schena et al., 1991, Proc. Natl. Acad. Sci. USA 88:0421).

Exemplary constitutive promoters include, but are not limited to: promoters from plant viruses, such as the 35S promoter from cauliflower mosaic virus (CaMV), promoters from the rice actin gene, ubiquitin promoters, pEMU, MAS , maize H3 histone promoter and ALS promoter, the Xba1/NcoI fragment (or polynucleotides similar to the Xba1/NcoI fragment) of the European rape (Brassica napus) ALS3 structural gene 5' (International PCT Publication No. WO96/ 30530).

Furthermore, any tissue-specific or tissue-preferred promoter may be utilized in some embodiments of the invention. A plant transformed with a nucleic acid molecule comprising an encoding polynucleotide operably linked to a tissue-specific promoter can produce the product of said encoding polynucleotide exclusively or preferentially in specific tissues. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to: seed-preferred promoters, such as the promoter from the phaseolin gene; leaf-specific and light-inducible promoters, such as those from cab or rubisco; anther-specific Pollen-specific promoters, such as the promoter from LAT52; pollen-specific promoters, such as the promoter from Zm13; and microspore-preferred promoters, such as the promoter from apg.

Transformation: As used herein, the term "transformation" or "transduction" refers to the transfer of one or more nucleic acid molecules into a cell. A cell is "transformed" by a nucleic acid molecule transduced into the cell when the nucleic acid molecule is stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cell's genome, or by episomal replication. As used herein, the term "transformation" encompasses all techniques by which nucleic acid molecules can be introduced into such cells. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation (Fromm et al., 1986, Nature 319:791-3); lipofection (Felgner et al., 1987, Proc. Natl.Acad.Sci.USA 84:7413-7); Microinjection (Mueller et al., 1978, Cell 15:579-85); Agrobacterium-mediated transfer (Fraley et al., 1983, USA80:4803-7); direct DNA uptake; and microparticle bombardment (Klein et al., 1987, Nature 327:70).

Transgenic: Exogenous nucleic acid. In some examples, a transgene may be DNA encoding one or both strands of RNA capable of forming a dsRNA molecule comprising a polynucleotide complementary to a nucleic acid molecule present in a coleopteran pest. In another example, the transgene can be an antisense polynucleotide, wherein expression of the antisense polynucleotide inhibits expression of a target nucleic acid, thereby producing an RNAi phenotype. In yet other examples, a transgene can be a gene (eg, a herbicide tolerance gene, a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired agricultural trait). In these and other examples, a transgene can contain a regulatory element (eg, a promoter) operably linked to the polynucleotide encoding the transgene.

Vector: A nucleic acid molecule that is introduced into a cell, eg, to produce a transformed cell. A vector may contain genetic elements that allow it to replicate in a host cell, such as an origin of replication. Examples of vectors include, but are not limited to: plasmids, cosmids, bacteriophages, or viruses that carry exogenous DNA into cells. A vector may also include one or more genes, including genes for the production of antisense molecules and/or selectable marker genes, as well as other genetic elements known in the art. A vector can transduce, transform or infect a cell, thereby causing the cell to express the nucleic acid molecule and/or the protein encoded by the vector. The carrier optionally includes substances that assist the nucleic acid molecule in achieving entry into the cell (eg, liposomes, protein coatings, etc.).

Yield: A stabilized yield of about 100% or greater relative to the yield of a test variety grown at the same growing location at the same time and under the same conditions. In particular embodiments, "increased yield" or "increased yield" means relative to the yield in a plant containing a substantial density of crop-damaging Coleopteran pests that are targeted by the compositions and methods herein. Yield of test varieties grown at the same growing location at the same time and under the same conditions, cultivars having a stabilized yield of 105% or greater.

Unless specifically stated or implied, the terms "a", "an" and "the/the" as used herein mean "at least one".

Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology can be found in publications such as: Lewin's Genes X , Jones & Bartlett Publishers, 2009 (ISBN 100763766321); Krebs et al. (editors), The Encyclopedia of Molecular Biology , Blackwell Science Ltd., 1994 (ISBN 0 -632-02182-9); and Meyers RA (editor), Molecular Biology and Biotechnology: A Comprehensive Desk Reference , VCH Publishers, Inc., 1995 (ISBN

1-56081-569-8). All percentages are by weight and all solvent mixture proportions are by volume unless otherwise indicated. All temperatures are in degrees Celsius.

IV. Nucleic acid molecules comprising insect pest sequences

A. Overview

Described herein are nucleic acid molecules useful for controlling insect pests. In some examples, the insect pest is a Coleopteran insect pest (eg, a Coleopteran pest in the genus Chrysoopteran). The described nucleic acid molecules include target polynucleotides (eg, native genes and non-coding polynucleotides), dsRNA, siRNA, shRNA, hpRNA, and miRNA. For example, in some embodiments dsRNA, siRNA, miRNA, shRNA and/or hpRNA molecules are described that are specifically complementary to all or part of one or more natural nucleic acids in a coleopteran pest. In these and additional embodiments, the one or more natural nucleic acids may be one or more target genes, the products of which may, for example and without limitation, participate in metabolic processes, or participate in larval development. A nucleic acid molecule described herein, when introduced into a cell comprising at least one natural nucleic acid that is specifically complementary to said nucleic acid molecule, can initiate RNAi in the cell, thereby reducing or eliminating expression of said natural nucleic acid. In some examples, reducing or eliminating the expression of a target gene by means of a nucleic acid molecule that is specifically complementary to the target gene can result in a slowing or cessation of the growth, development, viability, and/or feeding of the pest.

In some embodiments, at least one target gene in an insect pest can be selected, wherein the target gene comprises an spt6 polynucleotide. In a specific example, a target gene in a Coleopteran pest is selected, wherein the target gene comprises a polynucleotide selected from the group consisting of SEQ ID NO: 1 and 3-5.

In some embodiments, the target gene can be a nucleic acid molecule comprising a polynucleotide that can be back-translated in silico into a polypeptide comprising a contiguous amino acid sequence that is identical to The amino acid sequences of the protein products of the spt6 polynucleotides are at least about 85% identical (e.g., at least 84%, 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100% or 100% the same). The target gene may be any spt6 polynucleotide in an insect pest, the post-transcriptional inhibition of which polynucleotide has a detrimental effect on the growth, survival and/or viability of the pest, for example to provide a plant with a protective benefit against the pest. In a specific example, the target gene is a nucleic acid molecule comprising a polynucleotide that can be back-translated in silico into a polypeptide comprising a contiguous amino acid sequence identical to that of SEQ ID NO:2 The amino acid sequences are at least about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, about 99% identical, about 100% identical, or 100% identical.

According to the present invention there is provided a DNA whose expression produces an RNA molecule comprising a polynucleotide which is specifically complementary to all or part of a natural RNA molecule encoded by an encoding polynucleotide in an insect (eg Coleopteran) pest. In some embodiments, following ingestion of an expressed RNA molecule by an insect pest, downregulation of the encoding polynucleotide in cells of the pest can be obtained. In a particular embodiment, downregulation of an encoding polynucleotide in a pest cell can be obtained. In a specific embodiment, downregulation of an encoding polynucleotide in cells of an insect pest has deleterious effects on the growth and/or development of the pest.

In some embodiments, target polynucleotides include transcribed non-coding RNAs (such as 5'UTR, 3'UTR), splice leader sequences, introns, terminal introns (e.g., 5' UTR RNA), donatron (eg, non-coding RNA required to provide a trans-spliced donor sequence), and other non-coding transcribed RNAs that target insect pest genes. Such polynucleotides may be derived from both monocistronic and polycistronic genes.

Accordingly, also described herein in conjunction with some embodiments are iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) comprising at least one polynucleotide that interacts with a target nucleic acid in an insect (e.g., Coleopteran) pest All or part of the specific complementarity. In some embodiments, an iRNA molecule can comprise an iRNA that is complementary to all or part of a plurality of target nucleic acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more target nucleic acids). one or more polynucleotides. In specific embodiments, iRNA molecules can be produced in vitro, or in vivo by genetically modifying organisms such as plants or bacteria. Also disclosed are cDNAs that can be used to generate dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules and/or hpRNA molecules that are specifically complementary to all or part of a target nucleic acid in an insect pest. Recombinant DNA constructs for use in achieving stable transformation of specific host targets are further described. The transformed host target can express effective levels of dsRNA, siRNA, miRNA, shRNA and/or hpRNA molecules from the recombinant DNA construct. Accordingly, a plant transformation vector is also described comprising at least one polynucleotide operably linked to a heterologous promoter functional in a plant cell, wherein said one or more polynucleotides Expression produces an RNA molecule comprising a stretch of contiguous nucleobases that is specifically complementary to all or a portion of a target nucleic acid in an insect pest.

In particular examples, nucleic acid molecules useful for insect (e.g., Coleopteran) pests can include: all or part of a natural nucleic acid isolated from an organism of the genus Chrysophyll, comprising an spt6 polynucleotide (e.g., SEQ ID NO: 1 and any of 3-5); when expressed, DNA that produces an RNA molecule comprising a polynucleotide that is specifically complementary to all or part of a natural RNA molecule encoded by spt6; iRNA molecules (such as dsRNA, siRNA, miRNA, shRNA and hpRNA) of at least one polynucleotide specifically complementary to all or part of spt6; can be used to generate dsRNA molecules, siRNA molecules, miRNA specifically complementary to all or part of spt6 cDNA molecules, shRNA molecules and/or hpRNA molecules; and recombinant DNA constructs for use in achieving stable transformation of specific host targets, wherein the transformed host targets comprise one or more of the aforementioned nucleic acid molecules.

B. Nucleic acid molecules

In particular, the present invention provides iRNA (such as dsRNA, siRNA, miRNA, shRNA and hpRNA) molecules that inhibit the expression of target genes in cells, tissues or organs of insect (such as Coleoptera) pests; and those capable of being expressed as iRNA in cells or microorganisms A DNA molecule that inhibits the expression of a target gene in a cell, tissue or organ of an insect pest.

Some embodiments of the present invention provide an isolated nucleic acid molecule comprising at least one (eg, one, two, three or more) polynucleotides selected from the group consisting of: SEQ ID NO: 1; The complement of SEQ ID NO:1; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1 (e.g. any one of SEQ ID NOs:3-5); at least 15 contiguous cores of SEQ ID NO:1 The complementary sequence of a fragment of nucleotides; a naturally-encoded polynucleotide of an organism of the genus Chrysophyll (such as WCR) comprising any one of SEQ ID NOs: 3-5; a polynucleotide of a naturally-encoded organism of the genus Chrysophyll Complementary sequence of the naturally encoded polynucleotide comprising any one of SEQ ID NO: 3-5; a fragment of at least 15 contiguous nucleotides of a naturally encoded polynucleotide of an organism of the genus Chrysophyll, the naturally encoded polykaryotic The nucleotides comprise any one of SEQ ID NOs: 3-5; and the complement of a fragment of at least 15 contiguous nucleotides of a naturally-encoded polynucleotide of an organism of the genus Chrysophyll, the naturally-encoded polynucleotide comprising SEQ ID NO: Any one of ID NO:3-5.

In specific embodiments, exposure to or ingestion of an iRNA transcribed from the isolated polynucleotide by an insect (eg, Coleopteran) pest inhibits growth, development and/or feeding of the pest. In some embodiments, insect contact or ingestion occurs via feeding on plant material comprising the iRNA. In some embodiments, insect contact or ingestion occurs via spraying a plant containing the insect with a composition comprising the iRNA.

In some embodiments, an isolated nucleic acid molecule of the present invention may comprise at least one (eg, one, two, three or more) polynucleotides selected from the group consisting of: SEQ ID NO: 81; SEQ ID NO: 81; Complementary sequence of NO:81; SEQ ID NO:82; Complementary sequence of SEQ ID NO:82; SEQ ID NO:83; Complementary sequence of SEQ ID NO:83; SEQ ID NO:84; Complementary sequence of SEQ ID NO:84 ; a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; the complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; A native coding sequence of an organism of the genus comprising any one of SEQ ID NOs: 81-84; a complementary sequence of a native coding sequence of an organism of the genus Chrysophyll, said native coding sequence comprising any of SEQ ID NOs: 81-84 A fragment of at least 15 contiguous nucleotides of a native coding sequence of an organism of the genus Chrysophyll, said native coding sequence comprising any one of SEQ ID NOs: 81-84; and an organism of the genus Chrysophyll The complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence comprising any one of SEQ ID NOs: 81-84.

In specific embodiments, exposure to or ingestion of the isolated polynucleotide by a Coleopteran pest inhibits growth, development and/or feeding of the pest.

In certain embodiments, the dsRNA molecule provided herein comprises a polynucleotide complementary to a transcript from a target gene comprising SEQ ID NO: 1 or a fragment thereof, which inhibits the target gene in an insect pest from causing the pest The reduction or removal of polypeptide or polynucleotide agents necessary for the growth, development or other biological functions of the human body. The selected polynucleotide may exhibit about 80% to about 100% sequence identity to any of the following: SEQ ID NO: 1, contiguous fragments of SEQ ID NO: 1, and the complement of any of the foregoing. For example, selected polynucleotides can exhibit 79%, 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88% %, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, About 99.5% or about 100% sequence identity: SEQ ID NO: 1 , contiguous fragments of SEQ ID NO: 1 (eg, SEQ ID NOs: 3-5), or the complement of any of the foregoing.

In some embodiments, a DNA molecule capable of being expressed in a cell or microorganism as an iRNA molecule to inhibit expression of a target gene may comprise a polynucleotide associated with a native polynucleotide present in one or more target insect pest species (e.g., a Coleopteran pest species). All or part of a single polynucleotide that is specifically complementary, or a DNA molecule can be a chimera constructed from multiple such specifically complementary polynucleotides.

In some embodiments, a nucleic acid molecule may comprise a first polynucleotide and a second polynucleotide separated by a "spacer sequence." A spacer sequence may be a region comprising any nucleotide sequence that facilitates secondary structure formation (where desired) between the first polynucleotide and the second polynucleotide. In one embodiment, the spacer sequence is part of the sense-encoding polynucleotide or the antisense-encoding polynucleotide of the mRNA. Alternatively, the spacer sequence may comprise any combination of nucleotides or homologues thereof capable of being covalently linked to a nucleic acid molecule. In some examples, the spacer sequence can be a loop or an intron (eg, as the ST-LS1 intron).

For example, in some embodiments, a DNA molecule can comprise a polynucleotide encoding one or more different iRNA molecules, wherein each of the different iRNA molecules comprises a first polynucleotide and a second polynucleotide Nucleotides, wherein the first polynucleotide and the second polynucleotide are complementary to each other. The first polynucleotide and the second polynucleotide may be linked by a spacer sequence within the RNA molecule. A spacer sequence may form part of said first polynucleotide or said second polynucleotide. Expression of an RNA molecule comprising said first nucleotide polynucleotide and said second nucleotide polynucleotide may be achieved by said first nucleotide polynucleotide and said second nucleotide polynucleotide The specific intramolecular base pairing of dsRNA molecules results in the formation of dsRNA molecules. The first polynucleotide or the second polynucleotide may be compatible with a polynucleotide (e.g., a target gene or a transcribed non-coding polynucleotide), which is native to an insect pest (e.g., a coleopteran pest), Its derivatives or their complementary polynucleotides are substantially the same.

A dsRNA nucleic acid molecule comprises a double strand of polymerized ribonucleotides and may comprise modifications to the phosphate-sugar backbone or nucleosides. Modifications in RNA structure can be tailored to enable specific inhibition to occur. In one embodiment, dsRNA molecules can be modified by ubiquitous enzymatic processes so that siRNA molecules can be generated. The enzymatic process can utilize RNase III enzymes in vitro or in vivo, such as DICER in eukaryotes. See Elbashir et al., (2001) Nature 411:494-8; and Hamilton and Baulcombe, (1999) Science 286(5441):950-2. DICER, or a functionally equivalent RNase III enzyme, cleaves larger dsRNA strands and/or hpRNA molecules into smaller oligonucleotides (e.g., siRNA), each of which is about 19 in length. -25 nucleotides. siRNA molecules generated by these enzymes have 2 to 3 nucleotide 3' overhangs, as well as a 5' phosphate terminus and a 3' hydroxyl terminus. The siRNA molecule generated by the RNase III enzyme unwinds in the cell and separates into single-stranded RNA. The siRNA molecules then specifically hybridize to the RNA transcribed from the target gene, and both RNA molecules are subsequently degraded by the intrinsic cellular RNA degradation machinery. This process results in efficient degradation or removal of the RNA encoded by the target gene in the target organism. The result is post-transcriptional silencing of the targeted gene. In some embodiments, siRNA molecules produced from heterologous nucleic acid molecules by endogenous RNase III enzymes are effective in mediating downregulation of target genes in insect pests.

In some embodiments, a nucleic acid molecule can include at least one non-naturally occurring polynucleotide that can be transcribed into a single-stranded RNA molecule capable of forming a dsRNA molecule by intermolecular hybridization in vivo. Such dsRNAs typically self-assemble and can be provided in the nutritional source of insect (eg, Coleopteran) pests to achieve post-transcriptional repression of target genes. In these and additional embodiments, the nucleic acid molecule may comprise two different non-naturally occurring polynucleotides, wherein each polynucleotide is specifically complementary to a different target gene in the insect pest. When such a nucleic acid molecule is provided in the form of a dsRNA molecule to a coleopteran pest, for example, the dsRNA molecule inhibits the expression of at least two different target genes in the pest.

C. Obtaining Nucleic Acid Molecules

Nucleic acid molecules, such as iRNAs and DNA molecules encoding iRNAs, can be designed using a variety of polynucleotides in insect (eg, Coleopteran) pests as targets. However, selection of native polynucleotides is not a straightforward process. For example, only a few of the naturally occurring polynucleotides in coleopteran pests would be effective targets. It cannot be predicted with certainty whether a particular natural polynucleotide will be effectively downregulated by a nucleic acid molecule of the invention, or whether downregulation of a particular natural polynucleotide will adversely affect the growth, viability and/or development of an insect pest. The vast majority of natural coleopteran pest polynucleotides, such as ESTs isolated therefrom (eg, the coleopteran pest polynucleotides listed in US Pat. No. 7,612,194), do not adversely affect the growth and/or viability of the pests. It is also impossible to predict which of the natural polynucleotides that can have adverse effects on insect pests can be used in recombinant technology to express nucleic acid molecules complementary to such natural polynucleotides in host plants, and after the pests eat adversely affect it without causing harm to the host plant.

In some embodiments, a nucleic acid molecule (e.g., a dsRNA molecule to be provided in a host plant of an insect (e.g., Coleopteran) pest) is selected to target a cDNA encoding a protein necessary for the growth and/or development of the pest or protein moieties (such as polypeptides involved in metabolic or catabolic biochemical pathways, cell division, energy metabolism, digestion, host plant recognition, etc.). As described herein, the target pest organism ingests DNA containing one or more dsRNAs at least one segment of which is specifically complementary to at least one substantially identical segment of RNA produced in cells of the target pest organism. ) compositions that can result in death or other inhibition of the target. Polynucleotides (DNA or RNA) derived from insect pests can be used to construct pest-resistant plant cells. For example, a host plant of a coleopteran pest (eg, maize) can be transformed to contain one or more polynucleotides as provided herein derived from the coleopteran pest. The polynucleotide transformed into the host may encode one or more RNAs that form dsRNA structures in cells or biological fluids within the transformed host, thus, if the pest enters a vegetative relationship with the transgenic host or when the pest forms a vegetative relationship with the transgenic host Make the dsRNA available when related. This can result in the suppression of the expression of one or more genes in the pest cells and ultimately death or inhibition of pest growth or development.

Thus, in some embodiments, genes substantially involved in the growth and development of insect (eg, Coleopteran) pests are targeted. Other target genes used in the present invention may include, for example, those that play important roles in viability, movement, migration, growth, development, infectivity, and establishment of feeding sites of pests. Thus, the target gene can be a housekeeping gene or a transcription factor. In addition, the natural insect pest polynucleotides used in the present invention may also be derived from homologues (such as orthologs) of plant, viral, bacterial or insect genes, the function of which is known to those skilled in the art. and its polynucleotide can specifically hybridize with the target gene in the genome of the target pest. Methods for identifying homologues of genes having known nucleotide sequences by hybridization are known to those skilled in the art.

In some embodiments, the present invention provides methods for obtaining nucleic acid molecules comprising polynucleotides for the production of iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules. One such embodiment involves: (a) analyzing the expression, function and phenotype of one or more target genes in insect (e.g., Coleopteran) pests following dsRNA-mediated gene suppression; (b) using A cDNA or gDNA library is probed with a probe comprising all or part of a polynucleotide or a homologue thereof from a targeted pest that exhibits altered ( e.g. attenuated) growth or development phenotype; (c) identification of DNA clones that specifically hybridize to the probe; (d) isolation of DNA clones identified in step (b); (f) chemically synthesized genes or all of siRNA, miRNA, hpRNA, mRNA, shRNA or dsRNA or mostly.

In additional embodiments, the method for obtaining a nucleic acid fragment comprising a polynucleotide used to generate a majority of iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules comprises: (a) synthesizing a first oligonucleotide A nucleotide primer and a second oligonucleotide primer that are specifically complementary to a portion of a natural polynucleotide from the targeted insect (e.g., Coleopteran) pest; and (b) using the first oligonucleotide of step (a) The nucleotide primer and the second oligonucleotide primer amplify a cDNA or gDNA insert present in the cloning vector, wherein the amplified nucleic acid molecule comprises a majority of the siRNA, miRNA, hpRNA, mRNA, shRNA or dsRNA molecule.

Nucleic acids can be isolated, amplified or produced by a variety of methods. For example, iRNA (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) can be obtained by PCR amplifying target polynucleotides (e.g., target genes or transcribed target non-coding polynucleotides) or portions thereof derived from gDNA or cDNA libraries. )molecular. DNA or RNA can be extracted from the target organism, and nucleic acid libraries can be prepared from the DNA or RNA using methods known to those of ordinary skill in the art. The target gene can be PCR amplified and sequenced using a gDNA library or cDNA library generated from the target organism. Confirmed PCR products can be used as templates for in vitro transcription to generate sense and antisense RNA with minimal promoters. Alternatively, nucleic acid molecules can be synthesized by any of a number of techniques (see, e.g., Ozaki et al., (1992) Nucleic Acids Research, 20:5205-5214; and Agrawal et al., (1990) Nucleic Acids Research, 18:5419 -5423), including the use of an automated DNA synthesizer (eg, DNA/RNA Synthesizer Model 392 or 394 from P.E. Biosystems, Inc. (Foster City, Calif.)), using standard chemicals (such as phosphoramidite chemicals). See, eg, Beaucage et al., (1992) Tetrahedron, 48:2223-2311; US Patent Nos. 4,980,460, 4,725,677, 4,415,732, 4,458,066, and 4,973,679. Alternative chemistries such as phosphorothioates, phosphoroamidates, etc. that result in non-natural backbone groups may also be employed.

The RNA, dsRNA, siRNA, miRNA, shRNA or hpRNA molecules of the present invention can be produced chemically or enzymatically by those skilled in the art through manual or automated reactions, or in a Molecular polynucleotides are nucleic acid molecules that are produced in vivo in cells. RNA can also be produced by partial or complete organic synthesis, any modified ribonucleotides can be introduced by in vitro enzymatic or organic synthesis. RNA molecules can be synthesized by cellular RNA polymerase or phage RNA polymerase (eg, T3 RNA polymerase, T7 RNA polymerase, and SP6 RNA polymerase). Expression constructs useful for cloning and expressing polynucleotides are known in the art. See, eg, International PCT Publication No. WO97/32016, and US Patent Nos. 5,593,874, 5,698,425, 5,712,135, 5,789,214, and 5,804,693. RNA molecules synthesized chemically or by enzymatic synthesis in vitro can be purified prior to introduction into cells. For example, RNA molecules can be purified from a mixture by extraction with a solvent or resin, precipitation, electrophoresis, chromatography, or a combination of these means. Alternatively, RNA molecules synthesized chemically or by in vitro enzymatic synthesis can be used without or with minimal purification, eg, to avoid losses due to sample processing. RNA molecules can be dried for storage, or dissolved in an aqueous solution. The solution may contain buffers or salts to facilitate annealing and/or stabilization of the duplex strands of the dsRNA molecules.

In embodiments, dsRNA molecules can be formed by a single self-complementary RNA strand or by two complementary RNA strands. dsRNA molecules can be synthesized in vivo or in vitro. A cell's endogenous RNA polymerase can mediate transcription of one or both RNA strands in vivo, or a cloned RNA polymerase can be used to mediate transcription in vivo or in vitro. Post-transcriptional repression of target genes in insect pests by specific transcription in an organ, tissue or cell type of the host (e.g., by using a tissue-specific promoter); stimulation of environmental conditions in the host (e.g., by use of a response to infection, stress, temperature, and/or chemical inducers); and/or engineer transcription at a certain developmental stage or age of the host (e.g., by using a developmental stage-specific promoter) , can be host-targeted. The RNA strands that form the dsRNA molecules (whether transcribed in vitro or in vivo) may or may not be polyadenylated and may or may not be able to be translated into polypeptides by the cell's translation machinery.

D. Recombinant Vector and Host Cell Transformation

In some embodiments, the present invention also provides a DNA molecule for introduction into a cell (eg, a bacterial cell, a yeast cell, or a plant cell), wherein the DNA molecule comprises a polynucleotide expressed as After the RNA is ingested by an insect (eg, Coleoptera) pest, it can achieve the suppression of the target gene in the cell, tissue or organ of the pest. Accordingly, some embodiments provide recombinant nucleic acid molecules comprising polynucleotides capable of being expressed in plant cells as iRNA (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) molecules to inhibit expression of target genes in insect pests. To initiate or enhance expression, such recombinant nucleic acid molecules may comprise one or more regulatory elements, which may be operably linked to a polynucleotide capable of being expressed as iRNA. Methods for expressing gene repressor molecules in plants are known and can be used to express polynucleotides of the invention. See, eg, International PCT Publication No. WO06/073727; and US Patent Publication No. 2006/0200878 Al).

In specific embodiments, a recombinant DNA molecule of the invention may comprise a polynucleotide encoding an RNA capable of forming a dsRNA molecule. Such recombinant DNA molecules may encode RNAs that form dsRNA molecules that, when ingested, are capable of inhibiting the expression of one or more endogenous target genes in insect (eg, Coleopteran) pest cells. In many embodiments, the transcribed RNA can form dsRNA molecules that can be provided in a stabilized form; for example, in the form of hairpin and stem-loop structures.

In some embodiments, one strand of the dsRNA molecule can be formed by transcription from a polynucleotide that is substantially homologous to a polynucleotide selected from the group consisting of: SEQ ID NO: 1; the complement of SEQ ID NO: 1; SEQ ID NO: 1; A fragment of at least 15 contiguous nucleotides of ID NO: 1 (eg, SEQ ID NO: 3-5); the complementary sequence of a fragment of at least 15 contiguous nucleotides of SEQ ID NO: 1; For example, a naturally-encoded polynucleotide of WCR) comprising any one of SEQ ID NOs: 3-5; the complement of a naturally-encoded polynucleotide of an organism of the genus Chrysophyte comprising SEQ ID NO: Any of 3-5; a fragment of at least 15 contiguous nucleotides of a naturally-encoded polynucleotide of an organism of the genus Chrysophyll, comprising any of SEQ ID NOs: 3-5 ; the complement of a fragment of at least 15 contiguous nucleotides of a natively encoded polynucleotide of an organism of the genus Chrysophyll, comprising any one of SEQ ID NOs: 3-5.

In some embodiments, one strand of the dsRNA molecule may be formed by transcription from a polynucleotide that is substantially homologous to a polynucleotide selected from the group consisting of: SEQ ID NOs: 3-5; the complement of either; a fragment of at least 15 contiguous nucleotides of SEQ ID NO: 1 ; and the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO: 1 .

In particular embodiments, recombinant DNA molecules encoding RNAs that form dsRNA molecules may comprise a coding region in which at least two polynucleotides are arranged such that one polynucleotide is in sense relative to at least one promoter. orientation, and the other polynucleotide is in an antisense orientation, wherein the sense polynucleotide and the antisense polynucleotide are connected by, for example, a spacer sequence of about five (about 5) to about one thousand (about 1000) nucleotides or connected. A spacer sequence can form a loop between the sense polynucleotide and the antisense polynucleotide. The sense polynucleotide or antisense polynucleotide can be substantially homologous to a target gene (eg, the spt6 gene comprising any of SEQ ID NOs: 1 and 3-5) or a fragment thereof. However, in some embodiments, recombinant DNA molecules can encode RNAs that form spacer-free dsRNA molecules. In embodiments, the sense-encoding polynucleotide and the antisense-encoding polynucleotide can be of different lengths.

By creating appropriate expression cassettes in the recombinant nucleic acid molecules of the invention, polynucleotides identified as having deleterious effects on insect pests or having plant protective effects with respect to pests can readily be incorporated into expressed dsRNA molecules. For example, such a polynucleotide can be expressed as a hairpin having a stem-loop structure by obtaining a polynucleotide corresponding to a target gene (for example, comprising any one of SEQ ID NOs: 1 and 3-5, and spt6 gene of a fragment of any of the foregoing); the polynucleotide is linked to a second segment spacer region that is not homologous to the first segment or Complementary; the linker is then attached to a third segment, wherein at least a portion of the third segment is substantially complementary to the first segment. Such constructs form a stem-loop structure by intramolecular base pairing of the first segment with the third segment, wherein the loop structure forms comprising the second segment. See, eg, US Patent Publication Nos. 2002/0048814 and 2003/0018993; and International PCT Publication Nos. WO94/01550 and WO98/05770. The dsRNA molecule can be produced, for example, in the form of a double-stranded structure such as a stem-loop structure (e.g., a hairpin), thereby enhancing targeting by co-expressing fragments of the target gene, e.g., on an additional plant-expressible cassette. Production of siRNA from natural insect (eg, Coleopteran) pest polynucleotides, which results in enhanced siRNA production, or mitigation of methylation to prevent transcriptional gene silencing of dsRNA hairpin promoters.

Certain embodiments of the invention include introducing (ie, transforming) recombinant nucleic acid molecules of the invention into plants to achieve insect (eg, Coleopteran) pest-inhibiting levels of expression of one or more iRNA molecules. A recombinant DNA molecule may, for example, be a vector, such as a linear or closed circular plasmid. A vector system may be a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the host genome. Additionally, the vector may be an expression vector. A nucleic acid of the invention may be suitably inserted into a vector, eg, under the control of a suitable promoter which is functional in one or more hosts to drive expression of the linked encoding polynucleotide or other DNA element. Many vectors are available for this purpose, and the choice of an appropriate vector will depend primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components depending on the function of each vector (eg, amplifying DNA or expressing DNA) and the particular host cell with which it is compatible.

To confer protection to transgenic plants against insect (eg, Coleopteran) pests, for example, the recombinant DNA can be transcribed into iRNA molecules (eg, RNA molecules forming dsRNA molecules) within the tissues or fluids of the recombinant plants. The iRNA molecule can comprise a polynucleotide that is substantially homologous and specifically hybridizable to a corresponding transcribed polynucleotide in an insect pest that can cause damage to a host plant species. The pest can be exposed to the iRNA molecule transcribed in the transgenic host plant cell, eg, by ingesting cells or fluids of the transgenic host plant comprising the iRNA molecule. Thus, in specific examples, expression of a target gene is suppressed by an iRNA molecule in a coleopteran pest that infects a transgenic host plant. In some embodiments, suppression of expression of a target gene in a target coleopteran pest protects the plant from attack by the pest.

In order for an iRNA molecule to be delivered to an insect pest in vegetative relationship with a plant cell that has been transformed with a recombinant nucleic acid molecule of the invention, the iRNA molecule needs to be expressed (ie, transcribed) in the plant cell. Accordingly, a recombinant nucleic acid molecule may comprise a polynucleotide of the invention operably linked to one or more regulatory elements, such as a heterologous promoter element that functions in a host cell, such as in which A bacterial cell in which a nucleic acid molecule is amplified, and a plant cell in which a nucleic acid molecule is to be expressed.

Promoters suitable for use in the nucleic acid molecules of the present invention include inducible, viral, synthetic or constitutive promoters, all of which are well known in the art. Non-limiting examples describing such promoters include U.S. Patent Nos. 6,437,217 (maize RS81 promoter), 5,641,876 (rice actin promoter), 6,426,446 (maize RS324 promoter), 6,429,362 (maize PR-1 promoter), 6,232,526 (maize A3 promoter), 6,177,611 (maize constitutive promoter), 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (CaMV 35S promoter), 6,433,252 (maize L3 oleosin promoter), 6,429,357 (rice actin promoter) and rice actin 2 intron), 6,294,714 (light-inducible promoter), 6,140,078 (salt-inducible promoter), 6,252,138 (pathogen-inducible promoter), 6,175,060 (phosphate-deficiency-inducible promoter), 6,388,170 (bidirectional promoter), 6,635,806 (gamma-coixin (coixin) promoter), and US Patent Publication No. 2009/757,089 (maize chloroplast aldolase promoter). Additional promoters include the nopaline synthase (NOS) promoter (Ebert et al., (1987) Proc. Natl. Acad. Sci. USA 84(16):5745-9) and the octopine synthase (OCS) promoter (both are carried on the tumor-inducing plasmid of Agrobacterium tumefaciens); cauliflower mosaic virus group promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., (1987) Plant Mol .Biol.9:315-24); CaMV 35S promoter (Odell et al., (1985) Nature 313:810-2); Scrophulariae mosaic virus 35S-promoter (Walker et al., (1987) Proc.Natl .Acad.Sci.USA84(19):6624-8); Sucrose synthase promoter (Yang and Russell, (1990) Proc.Natl.Acad.Sci.USA 87:4144-8); R gene complex promoter (Chandler et al., (1989) Plant Cell 1:1175-83); Chlorophyll a/b binding protein gene promoter; CaMV 35S (US Patent Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196); 5,378,619); the PC1SV promoter (U.S. Patent No. 5,850,019); the SCP1 promoter (U.S. Patent No. 6,677,503); and the AGRtu.nos promoter (GenBank ^TM Accession No. V00087; Depicker et al., (1982) J.Mol.Appl.Genet .1:561-73; Bevan et al. (1983) Nature 304:184-7).

In particular embodiments, nucleic acid molecules of the invention comprise a tissue-specific promoter, such as a root-specific promoter. A root-specific promoter drives expression of an operably linked encoding polynucleotide exclusively or preferentially in root tissue. Examples of root-specific promoters are known in the art. See, eg, US Patent Nos. 5,110,732, 5,459,252, and 5,837,848; Opperman et al., (1994) Science 263:221-3; and Hirel et al., (1992) Plant Mol. Biol. 20:207-18. In some embodiments, polynucleotides or fragments for coleopteran pest control according to the present invention can be cloned between two root-specific promoters relative to the polynucleotide or The fragments are oriented in the opposite direction of transcription, operable in the transgenic plant cell, and expressed in the transgenic plant cell to produce therein RNA molecules that can then form dsRNA molecules, as described above. Insect pests can ingest iRNA molecules expressed in plant tissues, thereby suppressing the expression of target genes.

Additional regulatory elements that may optionally be operably linked to the nucleic acid include a 5'UTR positioned between the promoter element and the encoding polynucleotide to serve as a translation leader element. Translation leader elements are present in fully processed mRNA and can affect the processing of the primary transcript and/or the stability of the RNA. Examples of translation leader elements include maize and petunia heat shock protein leaders (US Pat. No. 5,362,865), plant viral coat protein leaders, plant ribulose diphosphate carboxylase (rubisco) leaders, and the like. See, eg, Turner and Foster, (1995) Molecular Biotech. 3(3):225-36. Non-limiting examples of 5'UTRs include GmHsp (US Patent No. 5,659,122), PhDnaK (US Patent No. 5,362,865), AtAnt1, TEV ( Carrington and Freed, (1990) J. Virol. 64:1590-7) and AGRtunos (GenBank ^™ Accession No. V00087; Bevan et al., (1983) Nature 304:184-7).

Additional regulatory elements that may optionally be operably linked to the nucleic acid also include 3' untranslated elements, 3' transcription termination regions or polyadenylation regions. These are genetic elements located downstream of polynucleotides, including polynucleotides that provide polyadenylation signals and/or other regulatory signals capable of affecting transcription or mRNA processing. Polyadenylation signals function in plants to cause the addition of polyadenylated nucleotides to the 3' end of mRNA precursors. Polyadenylation elements can be derived from a variety of plant genes or T-DNA genes. A non-limiting example of a 3' transcription termination region is the nopaline synthase 3' region (nos 3'; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803-7). An example of using a different 3' untranslated region is provided in Ingelbrecht et al. (1989) Plant Cell 1:671-80. Non-limiting examples of polyadenylation signals include the signal from the pea (Pisum sativum) RbcS2 gene (Ps.RbcS2-E9; Coruzzi et al., (1984) EMBO J.3:1671-9) and AGRtu.nos ( GenBank ^™ Accession No. E01312).

Some embodiments may include a plant transformation vector comprising an isolated and purified DNA molecule comprising at least one of the aforementioned regulatory elements operably linked to one or more polynucleotides of the invention By. The one or more polynucleotides, when expressed, generate one or more iRNA molecules comprising a polynucleotide specific for all or a portion of a natural RNA molecule in an insect (e.g., Coleopteran) pest sexual complementarity. Accordingly, the one or more polynucleotides may comprise a segment encoding all or part of a polyribonucleotide present in the RNA transcript of the targeted coleopteran pest, and may comprise the target pest An inverted repeat of all or part of a transcript. Plant transformation vectors may contain polynucleotides that are specifically complementary to more than one target polynucleotide, thereby allowing the production of more than one dsRNA to suppress two or more in cells of one or more populations or species of target insect pests. gene expression. Segments of polynucleotides that are specifically complementary to polynucleotides present in different genes can be combined into a single composite nucleic acid molecule for expression in transgenic plants. Such segments may be contiguous, or separated by a spacer sequence.

In some embodiments, a plasmid of the invention already containing at least one polynucleotide of the invention may be modified by sequentially inserting an additional polynucleotide or polynucleotides into the same plasmid, wherein the additional The or more polynucleotides are operably linked to the same regulatory element as the original at least one polynucleotide. In some embodiments, nucleic acid molecules can be designed to inhibit multiple target genes. In some embodiments, multiple genes to be inhibited can be obtained from the same insect (eg, Coleopteran) pest species, which can enhance the effectiveness of the nucleic acid molecule. In other embodiments, the genes can be derived from different insect pests, which can broaden the range of pests against which one or more agents are effective. Polycistronic DNA elements can be engineered when targeting multiple genes for repression or a combination of expression and repression.

A recombinant nucleic acid molecule or vector of the invention may comprise a selectable marker that confers a selectable phenotype on a transformed cell, such as a plant cell. Selectable markers can also be used to select plants or plant cells comprising recombinant nucleic acid molecules of the invention. The markers may encode biocide resistance, antibiotic resistance (e.g., kanamycin, geneticin (G418), bleomycin, hygromycin, etc.), or herbicide tolerance (e.g., glyphosate Wait). Examples of selectable markers include, but are not limited to: the neo gene encoding kanamycin resistance and which can be selected using kanamycin, G418, etc.; the bar gene encoding bialaphos resistance; encoding glyphosate tolerance the mutated EPSP synthase gene for bromoxynil; the nitrilase gene that confers resistance to bromoxynil; the mutated acetolactate synthase (ALS) gene that confers imidazolinone or sulfonylurea resistance; and the methotrexate resistance DHFR gene . A variety of selectable markers are available which confer resistance to ampicillin, bleomycin, chloramphenicol, gentamicin, hygromycin, kanamycin, lincomycin, methotrexate, grass Resistance to butylphosphine, puromycin, spectinomycin, rifampicin, streptomycin and tetracycline. Examples of such selectable markers are exemplified in, eg, US Patent Nos. 5,550,318, 5,633,435, 5,780,708 and 6,118,047.

A recombinant nucleic acid molecule or vector of the invention may also comprise a selectable marker. Expression can be monitored using a selectable marker. Exemplary selectable markers include: β-glucuronidase or uidA gene (GUS), the enzyme (Jefferson et al., (1987) Plant Mol.Biol.Rep.5:387 of its encoding known various chromogenic substrates) -405); the R-locus gene, which encodes a product that regulates the production of anthocyanin pigments (red) in plant tissues (Dellaporta et al., (1988) "Molecular cloning of the maize R-nj allele by transposon tagging with Ac", In ^18th Stadler Genetics Symposium , edited by P. Gustafson and R. Appels, NewYork: Plenum, pp. 263-82); β-lactamase gene (Sutcliffe et al., (1978) Proc.Natl.Acad.Sci.USA 75:3737-41); genes encoding enzymes known to be various chromogenic substrates (eg PADAC, a chromogenic cephalosporin); luciferase gene ( Ow et al., (1986) Science 234:856-9); the xylE gene, which encodes a catechol dioxygenase that converts chromogenic catechols (Zukowski et al., (1983) Gene 46(2-3) :247-55); the amylase gene (Ikatu et al., (1990) Bio/Technol.8:241-2); the tyrosinase gene, which encodes the ability to oxidize tyrosine to DOPA and dopaquinone (its and α-galactosidase.

In some embodiments, in methods for creating transgenic plants and expressing heterologous nucleic acids in plants, recombinant nucleic acid molecules as described hereinbefore may be used to produce genes exhibiting susceptibility to insect (e.g., Coleopteran) pests. Reduced transgenic plants. Plant transformation vectors can be prepared, for example, by inserting nucleic acid molecules encoding iRNA molecules into plant transformation vectors and then introducing these into plants.

Suitable methods for transforming host cells include any method that can introduce DNA into the cell, such as by transformation of protoplasts (see, e.g., U.S. Pat. No. 5,508,184), by desiccation/inhibition-mediated DNA uptake (see, e.g., Potrykus et al., ( 1985) Mol. Gen. Genet. 199:183-8), by electroporation (see, e.g., U.S. Pat. No. 5,384,253), by agitation with silicon carbide fibers (see, e.g., U.S. Pat. Transformation (see, e.g., U.S. Pat. Nos. 5,563,055, 5,591,616, 5,693,512, 5,824,877, 5,981,840, and 6,384,301) and by accelerating DNA-coated particles (see, e.g., U.S. Pat. Techniques that are particularly useful for transforming maize are described, for example, in US Patent Nos. 7,060,876 and 5,591,616, and International PCT Publication No. WO95/06722. By applying techniques such as these, cells of almost any species can be stably transformed. In some embodiments, the transforming DNA is integrated into the genome of the host cell. In the case of multicellular species, transgenic cells can be regenerated into transgenic organisms. Any of these techniques can be used to generate a transgenic plant, eg, a transgenic plant comprising in its genome one or more nucleic acids encoding one or more iRNA molecules.

The most widely used method for introducing expression vectors into plants is based on the natural transformation system of Agrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry the genes responsible for the genetic transformation of plants. Ti (tumor-inducing) plasmids contain a large segment called T-DNA that transfers to transformed plants. Another segment of the Ti plasmid, the Vir region, is responsible for T-DNA transfer. The T-DNA region is bordered by terminal repeats. In the modified binary vector, the tumor-inducing gene has been deleted, and the function of the Vir region is used to transfer the foreign DNA bordered by T-DNA border elements. The T-region may also contain selectable markers for efficient recovery of transgenic cells and plants, as well as multiple cloning sites for insertion of transfer polynucleotides such as dsRNA-encoding nucleic acids.

Thus, in some embodiments, the plant transformation vector is derived from the Ti plasmid of A. tumefaciens (see, e.g., U.S. Pat. plasmid. Additional plant transformation vectors include, for example but not limited to, those described by Herrera-Estrella et al., (1983) Nature 303:209-13; Bevan et al., (1983) Nature 304:184-7; Klee et al., (1985) Bio/Technol. 3:637-42; and those described in European Patent No. EP 0 120 516, and those derived from any of the aforementioned vectors. Other bacteria that naturally interact with plants, such as Sinorhizobium, Rhizobium, and Mesorhizobium, can be modified to mediate gene transfer. These plant-associated commensal bacteria can be rendered competent for gene transfer by obtaining both a disarmed Ti plasmid and an appropriate binary vector.

After providing exogenous DNA to recipient cells, transformed cells are typically identified for further culture and plant regeneration. To improve the ability to identify transformed cells, the skilled artisan may desire to employ selectable or screenable marker genes as set forth previously, where the transformation vector is used to generate transformants. Where a selectable marker is used, transformed cells are identified within a population of potentially transformed cells by exposing the cells to one or more selective agents. Where a selectable marker is used, cells can be screened for the desired marker gene trait.

Cells that survive exposure to the selection agent, or that have scored positive in a screening assay, can be cultured in a medium that supports plant regeneration. In some embodiments, any suitable plant tissue culture medium (eg, MS medium and N6 medium) can be modified by the inclusion of additional substances, such as growth regulators. Tissue can be maintained on basal medium with growth regulators until sufficient tissue is available to initiate plant regeneration efforts, or after repeated rounds of manual selection until tissue morphology is suitable for regeneration (e.g. , at least 2 weeks), and then transferred to a medium conducive to shoot formation. Cultures were transferred periodically until sufficient shoot formation had occurred. Once the shoots have formed, transfer them to a medium that is good for root formation. Once sufficient roots have developed, the plants can be transferred to soil for further growth and maturation.

To confirm the presence of a nucleic acid molecule of interest (eg, DNA encoding one or more iRNA molecules that inhibit expression of a target gene in a coleopteran pest) in regenerated plants, various assays can be performed. Such assays include, for example: molecular biological assays, such as Southern and Northern blots, PCR and nucleic acid sequencing; biochemical assays, such as detection of the presence or absence of protein products, e.g. by immunological means (ELISA and/or Western blot) or with the aid of enzymes Function; plant part assays, such as leaf or root assays; and analysis of phenotypes in regenerated whole plants.

Integration events can be analyzed, for example, by PCR amplification using, for example, oligonucleotide primers specific for the nucleic acid molecule of interest. PCR genotyping should be understood to include, but is not limited to, polymerase chain reaction (PCR) amplification of gDNA derived from isolated host plant calli predicted to contain the gene of interest integrated into the genome. Nucleic acid molecules, followed by standard cloning and sequence analysis of PCR amplification products. Methods for PCR genotyping are well described (e.g. Rios, G. et al. (2002) Plant J. 32:243-53) and can be applied to gDNA derived from any plant species (e.g. maize) or tissue type , including gDNA derived from cell culture.

Transgenic plants formed using Agrobacterium-dependent transformation methods typically contain a single recombinant DNA inserted into one chromosome. This single polynucleotide of recombinant DNA is referred to as a "transgenic event" or "integration event". Such transgenic plants are heterozygous for the inserted exogenous polynucleotide. In some embodiments, T1 seeds that are homozygous for the transgene can be obtained by sexually mating (selfing) _an independent, segregating transgenic plant containing a single exogenous gene to itself (e.g., a _T0 plant). transgenic plants. _A quarter of the T1 seeds produced will be homozygous for the transgene. Plants produced by germinating T1 _seeds can be used to test for heterozygosity, typically using SNP assays or thermal amplification assays, allowing the distinction between heterozygotes and homozygotes (ie, zygosity assays).

In specific embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more, different iRNAs having an insect (e.g., Coleopteran) pest-inhibiting effect are produced in a plant cell molecular. An iRNA molecule (eg, a dsRNA molecule) can be expressed from multiple nucleic acids introduced into different transformation events, or from a single nucleic acid introduced into a single transformation event. In some embodiments, multiple iRNA molecules are expressed under the control of a single promoter. In other embodiments, multiple iRNA molecules are expressed under the control of multiple promoters. A single iRNA molecule may be expressed comprising multiple polynucleotides each linked to a different locus within one or more insect pests in different populations of the same insect pest species or in different insect pest species (eg, the locus defined by SEQ ID NO: 1) homologous.

In addition to directly transforming plants with recombinant nucleic acid molecules, transgenic plants can be prepared by crossing a first plant having at least one transgenic event with a second plant lacking such an event. For example, a recombinant nucleic acid molecule comprising a polynucleotide encoding an iRNA molecule can be introduced into a first plant line amenable to transformation to produce a transgenic plant which can be crossed with a second plant line such that the polynucleotide encoding the iRNA molecule Introgression into a second plant line.

In some aspects, seeds and commercial products produced by transgenic plants derived from transformed plant cells are included, wherein the seeds or commercial products comprise detectable amounts of nucleic acids of the invention. In some embodiments, such commercial products can be produced, for example, by obtaining transgenic plants and preparing food or feed therefrom. Commercial products comprising one or more of the polynucleotides of the invention include, for example but not limited to: meal, oil, ground or whole grains or seeds of plants, and Any meal, oil, or ground or whole grain of any food product of one or more of the recombinant plants or seeds. The detection of one or more of the polynucleotides of the invention in one or more commercial or commercial products substantially demonstrates that the commercial or commercial products are produced for the purpose of controlling insect (e.g. Coleopteran) pests Transgenic plants designed to express one or more of the iRNA molecules of the invention are produced.

In some embodiments, a transgenic plant or seed comprising a nucleic acid molecule of the present invention may also comprise at least one other transgenic event in its genome, including but not limited to: from its transcriptional target coleopteran pest and identified by SEQ ID NO: 1 Transgenic events of iRNA molecules of defined loci different loci, such as one or more loci selected from the group consisting of: Caf1-180 (US Patent Application Publication No. 2012/0174258), VatpaseC (US Patent Application Publication No. 2012/0174259), Rho1 (US Patent Application Publication No. 2012/0174260), VatpaseH (US Patent Application Publication No. 2012/0198586), PPI-87B (US Patent Application Publication No. 2013/0091600), RPA70 (U.S. Patent Application Publication No. 2013/0091601), RPS6 (U.S. Patent Application Publication No. 2013/0097730), ROP (U.S. Patent Application No. 14/577,811), RNA polymerase II140 (U.S. Patent Application No. 14/577,854), Dre4 ( U.S. Patent Application No. 14/705,807), ncm (U.S. Patent Application No. 62/095487), COPIα (U.S. Patent Application No. 62/063,199), COPIβ (U.S. Patent Application No. 62/063,203), COPIγ (U.S. Patent Application No. 62/ 063,192), COIPδ (US Patent Application No. 62/063,216), RNA Polymerase I1 (US Patent Application No. 62/133214), RNA Polymerase II-215 (US Patent Application No. 62/133,202), RNA Polymerase 33 (US Patent Application No. Patent Application No. 62/133210) and histone chaperone spt5 (US Patent Application No. 62/168613); transgenes from iRNA molecules whose transcription targets genes in organisms other than coleopteran pests, such as plant parasitic nematodes events; genes encoding insecticidal proteins (e.g., Bacillus thuringiensis insecticidal proteins); herbicide tolerance genes (e.g., genes providing tolerance to glyphosate); and contributing to desired phenotypes in transgenic plants ( genes such as increased yield, altered fatty acid metabolism, or restoration of cytoplasmic male sterility). In particular embodiments, polynucleotides encoding iRNA molecules of the invention can be combined with other insect control and disease control traits in plants to achieve the desired trait of enhanced control of plant disease and insect damage. Combining insect control traits employing unique modes of action can provide protected plants with superior durability over plants containing a single control trait due to, for example, a reduced probability that resistance to the one or more traits will develop in the field. transgenic plants.

V. Target gene suppression in insect pests

A. Overview

In some embodiments of the invention, an insect (eg, Coleopteran) pest may be provided with at least one nucleic acid molecule useful for controlling the insect pest, wherein the nucleic acid molecule causes RNAi-mediated gene silencing in the pest. In particular embodiments, iRNA molecules (eg, dsRNA, siRNA, miRNA, shRNA, and hpRNA) can be provided to coleopteran pests. In some embodiments, a nucleic acid molecule useful for controlling an insect pest can be provided to a pest by contacting the nucleic acid molecule with the pest. In these and additional embodiments, nucleic acid molecules useful for controlling an insect pest can be provided in a feeding matrix (eg, a nutritional composition) for the pest. In these and additional embodiments, the nucleic acid molecule useful for controlling the pest may be provided by ingesting plant material ingested by the insect pest comprising the nucleic acid molecule. In certain embodiments, the nucleic acid molecule is present in the plant material by expressing the recombinant nucleic acid introduced into the plant material, e.g., by transforming a plant cell with a vector comprising the recombinant nucleic acid, and then regenerating the plant from the transformed plant cell materials or whole plants.

B. RNAi-mediated target gene suppression

In some embodiments, the invention provides iRNA molecules (e.g., dsRNA, siRNA, miRNA, shRNA, and hpRNA) that can be designed to target insect pests (e.g., pests of the order Coleoptera (e.g., WCR, NCR, and SCR). An essential natural polynucleotide (eg, an essential gene) in the transcriptome of ), eg, by designing an iRNA molecule with at least one strand comprising a polynucleotide that is specifically complementary to the target polynucleotide. The sequence of the iRNA molecule so designed may be identical to the sequence of the target polynucleotide, or may incorporate mismatches that do not prevent specific hybridization between the iRNA molecule and its target polynucleotide.

The iRNA molecules of the invention can be used in methods for gene suppression in insect (e.g., Coleopteran) pests, thereby reducing the likelihood of damage caused by the pest to plants (e.g., protected transformed plants comprising the iRNA molecule) level or incidence. As used herein, the term "gene suppression" refers to any well-known method for reducing the level of protein produced as a result of the transcription of a gene into mRNA and the subsequent translation of the mRNA, including the reduction of a protein expressed by a gene or an encoding polynucleotide, Includes post-transcriptional repression of expression and transcriptional repression. Post-transcriptional repression is mediated by specific homology between all or part of the mRNA transcribed from a gene targeted for repression and the corresponding iRNA molecule used for repression. Furthermore, post-transcriptional repression refers to a substantial and measurable decrease in the amount of mRNA available in a cell for incorporation by ribosomes.

In some embodiments where the iRNA molecule is a dsRNA molecule, the enzyme DICER can cleave the dsRNA molecule into short siRNA molecules (approximately 20 nucleotides in length). Double-stranded siRNA molecules generated by DICER activity on dsRNA molecules can be split into two single-stranded siRNAs: a "passenger strand" and a "guide strand". The passenger strand can be degraded, and the guide strand can be incorporated into RISC. Post-transcriptional repression occurs through specific hybridization of the guide strand to a specific complementary polynucleotide of the mRNA molecule, followed by cleavage by the enzyme Argonaute, the catalytic component of the RISC complex.

In some embodiments of the invention, any form of iRNA molecule can be used. Those of skill in the art will appreciate that dsRNA molecules are generally more stable than single-stranded RNA molecules during manufacture and during the step of providing an iRNA molecule to a cell, and are generally more stable in the cell. Thus, for example, while siRNA molecules and miRNA molecules may be equally effective in some embodiments, dsRNA molecules may be chosen for their stability.

In specific embodiments, nucleic acid molecules are provided comprising polynucleotides expressible in vitro to produce iRNA molecules that are identical to polynucleotides encoded by polynucleotides within the genome of insect (e.g., Coleopteran) pests. Nucleic acid molecules are substantially homologous. In certain embodiments, the in vitro transcribed iRNA molecule can be a stabilized dsRNA molecule comprising a stem-loop structure. Following exposure of an insect pest to an in vitro transcribed iRNA molecule, post-transcriptional repression of a target gene (eg, an essential gene) in the pest can occur.

In some embodiments of the invention, at least 15 contiguous nucleotides (e.g., at least 19 contiguous cores) comprising a polynucleotide are used in methods for post-transcriptional suppression of target genes in insect (e.g., Coleoptera) pests. nucleotides), wherein the polynucleotide is selected from the group consisting of: SEQ ID NO: 1; the complementary sequence of SEQ ID NO: 1; SEQ ID NO: 3; the complementary sequence of SEQ ID NO: 3; SEQ ID NO :4; the complement of SEQ ID NO:4; SEQ ID NO:5; the complement of SEQ ID NO:5; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; at least 15 of SEQ ID NO:1 The complement of a fragment of contiguous nucleotides; a native coding sequence of an organism of the genus Chrysophyll, comprising any one of SEQ ID NOs: 3-5; the complement of a native coding sequence of an organism of the genus Chrysophyll, wherein The native coding sequence comprises any one of SEQ ID NO:3-5; a fragment of at least 15 contiguous nucleotides of a native coding sequence of a Chrysophyll organism comprising SEQ ID NO:3- Any of 5; the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of an organism of the genus Chrysophyll, said native coding sequence comprising any of SEQ ID NOs: 3-5. In certain embodiments, at least about 80% (e.g., 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% , about 99%, about 100%, and 100%) identical expression of nucleic acid molecules. In these and additional embodiments, a nucleic acid molecule that specifically hybridizes to an RNA molecule present in at least one cell of an insect (eg, Coleopteran) pest can be expressed.

An important feature of some embodiments herein is that the RNAi post-transcriptional suppression system is able to tolerate sequence variations in the target gene that are expected to occur as a result of genetic mutation, strain polymorphism, or evolutionary divergence. The introduced nucleic acid molecule does not have to be absolutely homologous to the primary transcript or fully processed mRNA of the target gene, as long as the introduced nucleic acid molecule can specifically hybridize to the primary transcript or fully processed mRNA of the target gene. Additionally, the introduced nucleic acid molecule need not be full length relative to the primary transcript or fully processed mRNA of the target gene.

Inhibition of target genes using the iRNA technology of the invention is sequence specific; that is, genetic inhibition targets polynucleotides that are substantially homologous to one or more iRNA molecules. In some embodiments, inhibition can be performed using an RNA molecule comprising a polynucleotide whose nucleotide sequence is identical to that of a portion of the target gene. In these and additional embodiments, RNA molecules comprising polynucleotides with one or more insertions, deletions and/or point mutations relative to the target polynucleotide can be used. In particular embodiments, the iRNA molecule and a portion of the target gene may share, for example, at least from about 80%, at least from about 81%, at least from about 82%, at least from about 83%, at least from about 84%, at least from about 85%, at least from about 86%, at least from about 87%, at least from about 88%, at least from about 89%, at least from about 90%, at least from about 91%, at least from about 92%, at least from about 93% , at least from about 94%, at least from about 95%, at least from about 96%, at least from about 97%, at least from about 98%, at least from about 99%, at least from about 100%, and 100% sequence identity. Alternatively, the duplex region of the dsRNA molecule can specifically hybridize to a portion of the target gene transcript. In specifically hybridizable molecules, less than full-length polynucleotides exhibiting greater homology compensate for longer, less homologous polynucleotides. The polynucleotide in the duplex region of the dsRNA molecule that is identical to a portion of the target gene transcript can be at least about 25, 50, 100, 200, 300, 400, 500, or at least about 1000 bases in length. In some embodiments, polynucleotides of greater than 20 to 100 nucleotides may be used. In particular embodiments, polynucleotides greater than about 100-500 nucleotides may be used. In specific embodiments, depending on the size of the target gene, polynucleotides greater than about 500 to 1000 nucleotides may be used.

In certain embodiments, expression of a target gene in a pest (eg, Coleoptera) can be inhibited by at least 10%, at least 33%, at least 50%, or at least 80% in cells of the pest such that significant inhibition occurs. Significant inhibition refers to inhibition above a threshold that results in a detectable phenotype (e.g., growth arrest, feeding arrest, developmental arrest, induced death, etc.), or RNA corresponding to the target gene being suppressed and/or There is a detectable reduction in the gene product. While in certain embodiments of the invention suppression occurs in substantially all cells of the pest, in other embodiments suppression occurs only in a subset of cells expressing the target gene.

In some embodiments, transcriptional repression in the cell is mediated by the presence of a dsRNA molecule exhibiting substantial sequence identity to the promoter DNA or its complement, thereby achieving so-called "promoter transrepression" . Gene suppression can be effected against a target gene in an insect pest that can ingest or contact such dsRNA molecules (eg, by ingesting or contacting plant material containing the dsRNA molecules). The dsRNA molecules used in promoter transrepression may be specifically designed to inhibit or repress the expression of one or more homologous or complementary polynucleotides in insect pest cells. Post-transcriptional gene repression by antisense or sense oriented RNA to regulate gene expression in plant cells is disclosed in US Patent Nos. 5,107,065, 5,759,829, 5,283,184 and 5,231,020.

C. Expression of iRNA Molecules Provided to Insect Pests

iRNA molecules for RNAi-mediated gene suppression in insect (eg, Coleopteran) pests can be expressed in any of a number of in vitro or in vivo formats. The iRNA molecule can then be provided to an insect pest, for example, by contacting the iRNA molecule with the pest, or by causing the pest to ingest or otherwise internalize the iRNA molecule. Some embodiments include transformed host plants, transformed plant cells, and progeny of transformed plants of coleopteran pests. Transformed plant cells and transformed plants can be engineered to express one or more of the iRNA molecules, eg, under the control of a heterologous promoter, to provide a pest protective effect. Thus, when an insect pest consumes a transgenic plant or plant cell during feeding, the pest can ingest the iRNA molecule expressed in the transgenic plant or cell. The polynucleotides of the invention can also be introduced into a wide variety of prokaryotic and eukaryotic microbial hosts to produce iRNA molecules. The term "microorganism" includes prokaryotic and eukaryotic species, such as bacteria and fungi.

Modulating gene expression can include partial or complete repression of such expression. In another embodiment, a method for suppressing gene expression in an insect (e.g., Coleoptera) pest comprises providing in a tissue of a pest host a gene suppressive amount of a gene formed post-transcriptionally by a polynucleotide as described herein. At least one dsRNA molecule of which at least one segment is complementary to an mRNA in a cell of an insect pest. dsRNA molecules ingested by insect pests, including modified forms thereof such as siRNA, miRNA, shRNA or hpRNA molecules, can be combined with RNA transcribed from, for example, an spt6 DNA molecule comprising a polynucleotide selected from SEQ ID NO: 1 and 3-5. Molecular at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or about 100% the same. Accordingly, provided are isolated and substantially purified nucleic acid molecules useful in the preparation of dsRNA molecules, including but not limited to non-naturally occurring polynucleotides and recombinant DNA constructs, which when introduced into an insect pest suppress or inhibit endogenous Expression of a sex-encoding polynucleotide or a target-encoding polynucleotide.

Certain embodiments provide delivery systems for delivering iRNA molecules for post-transcriptional inhibition of one or more target genes in insect (eg, Coleopteran) plant pests and control of populations of said plant pests. In some embodiments, the delivery system comprises uptake of host transgenic plant cells or host cell contents comprising RNA molecules transcribed in the host cells. In these and additional embodiments, transgenic plant cells or transgenic plants are created that contain a recombinant DNA construct for providing a stabilized dsRNA molecule of the invention. Transgenic plant cells and transgenic plants comprising a nucleic acid encoding a particular iRNA molecule can be produced by constructing a molecule comprising an iRNA encoding the invention (e.g., a stabilized dsRNA) using recombinant DNA techniques (such basic techniques are well known in the art). A plant transformation vector of a polynucleotide of a molecule), thereby transforming a plant cell or plant, and thereby generating a transgenic plant cell or a transgenic plant containing a transcribed iRNA molecule.

To confer protection to transgenic plants against insect (eg Coleopteran) pests, recombinant DNA molecules can be transcribed, for example, into iRNA molecules, such as dsRNA molecules, siRNA molecules, miRNA molecules, shRNA molecules or hpRNA molecules. In some embodiments, RNA molecules transcribed from recombinant DNA molecules can form dsRNA molecules within tissues or fluids of recombinant plants. Such a dsRNA molecule may be comprised in a portion of a polynucleotide identical to a corresponding polynucleotide transcribed from DNA within an insect pest of the type that can infect a host plant. Expression of the target gene in the pest is suppressed by the dsRNA molecule, and suppression of expression of the target gene in the pest protects the transgenic plant from damage by the pest. Regulation of dsRNA molecules has been shown to apply to a variety of genes expressed in pests, including, for example, endogenous genes responsible for cell metabolism or cell transformation, including housekeeping genes; transcription factors; molt-related genes; and genes encoding genes involved in cell metabolism or normal growth. and other genes of developmental polypeptides.

For transcription from an in vivo transgene or expression construct, regulatory regions (eg, promoters, enhancers, silencers, and polyadenylation signals) may be used in some embodiments to transcribe one or more RNA strands. Thus, in some embodiments, polynucleotides used in the production of iRNA molecules may be operably linked to one or more promoter elements that are functional in a plant host cell, as previously set forth. The promoter may be an endogenous promoter that normally resides in the host genome. A polynucleotide of the invention under the control of an operably linked promoter element may be further flanked by additional elements that advantageously affect its transcription and/or the stability of the resulting transcript. Such elements can be located upstream of the operably linked promoter, downstream of the 3' end of the expression construct, and can be present both upstream of the promoter and downstream of the 3' end of the expression construct.

Some embodiments provide a method for alleviating damage to a host plant (e.g., a corn plant) caused by a plant-feeding insect (e.g., Coleoptera) pest, wherein the method comprises providing in the host plant an expression A transformed plant cell of at least one nucleic acid molecule, wherein said nucleic acid molecule, upon ingestion by said pest, acts to inhibit expression of a target polynucleotide in said pest, the inhibition of expression causing death and/or growth of said pest Slow down, thereby lessening the damage to the host plant caused by the pest. In some embodiments, the nucleic acid molecule comprises a dsRNA molecule. In these and additional embodiments, the nucleic acid molecules comprise dsRNA molecules each comprising more than one polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in a Coleopteran insect pest cell. In some embodiments, the nucleic acid molecule consists of a polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell.

In some embodiments, there is provided a method for increasing crop yield, wherein the method comprises introducing at least one nucleic acid molecule of the present invention into a corn plant; cultivating the corn plant to allow expression of an iRNA molecule comprising the nucleic acid , wherein expression of an iRNA molecule comprising said nucleic acid inhibits insect (eg, Coleopteran) pest damage and/or growth, thereby reducing or eliminating yield loss due to pest infestation. In some embodiments, the iRNA molecule is a dsRNA molecule. In these and additional embodiments, the nucleic acid molecules comprise dsRNA molecules each comprising more than one polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell. In some embodiments, the nucleic acid molecule comprises a polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in a Coleopteran pest cell.

In an alternative embodiment, there is provided a method for modulating the expression of a target gene in an insect (e.g., Coleoptera) pest comprising: transforming with a vector comprising a polynucleotide encoding at least one iRNA molecule of the invention A plant cell, wherein the polynucleotide is operably linked to a promoter and a transcription termination element; culturing the transformed plant cell under conditions sufficient to allow the development of a plant cell culture comprising a plurality of transformed plant cells; selecting for the transformed plant cell the transformed plant cell into which the polynucleotide is integrated into its genome; the transformed plant cell is screened for expression of the iRNA molecule encoded by the integrated polynucleotide; the transgenic plant cell expressing the iRNA molecule is selected; and the selected transgenic plant is then The cells are fed to the insect pest. Plants can also be regenerated from transformed plant cells expressing the iRNA molecule encoded by the integrated nucleic acid molecule. In some embodiments, the iRNA molecule is a dsRNA molecule. In these and additional embodiments, the nucleic acid molecules comprise dsRNA molecules each comprising more than one polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in an insect pest cell. In some embodiments, the nucleic acid molecule comprises a polynucleotide that specifically hybridizes to a nucleic acid molecule expressed in a Coleopteran pest cell.

The iRNA molecules of the invention can be incorporated into the seeds of a plant species (e.g., maize) as the expression product from a recombinant gene incorporated into the plant cell genome, or as a coating or seed treatment applied to the seed prior to planting. within. A plant cell containing a recombinant gene is considered a transgenic event. Also included in embodiments of the invention are delivery systems for delivering iRNA molecules to insect (eg, Coleopteran) pests. For example, the iRNA molecule of the present invention can be directly introduced into the cells of the pest. Methods for introduction may include mixing the iRNA directly with plant tissue from an insect pest host, and applying to the host plant tissue a composition comprising an iRNA molecule of the invention. For example, iRNA molecules can be sprayed onto the surface of a plant. Alternatively, iRNA molecules can be expressed by microorganisms, which can then be applied to the plant surface, or introduced into roots or shoots by physical means such as injection. As previously discussed, transgenic plants can also be genetically engineered to express at least one iRNA molecule in an amount sufficient to kill an insect pest known to infest the plant. iRNA molecules produced by chemical or enzymatic synthesis methods can also be formulated in a manner consistent with common agricultural practices and used as spray or bait products to control plant damage by insect pests. Formulations may contain suitable adhesives and wetting agents required for effective leaf coverage, as well as UV protectants to protect iRNA molecules (eg, dsRNA molecules) from UV damage. Such additives are commonly used in the biopesticide industry and are well known to those skilled in the art. Such applications may be combined with other spray insecticide applications (biologically based or otherwise) to increase the protection of plants against said pests.

All references cited herein, including publications, patents, and patent applications, are hereby incorporated by reference to the same extent as not inconsistent with explicit details of the present disclosure, and to the same extent as if individually Each reference is incorporated by reference as specifically and specifically indicated and is set forth in its entirety herein. The references discussed herein are provided solely for reference to their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention.

The following examples are provided to illustrate certain specific features and/or aspects. These examples should not be construed to limit the disclosure to the particular features or aspects described.

Example

Example 1: Materials and methods

Sample Preparation and Bioassays

use RNAi kit or T7 in vitro transcription kit synthesized and purified a variety of dsRNA molecules (including those corresponding to spt6-1 reg1 (SEQ ID NO:3), spt6-1 v1 (SEQ ID NO:4) and spt6-1 v2 (SEQ ID NO: 5) those molecules). Purified dsRNA molecules were prepared in TE buffer, which consisted of a control treatment for all bioassays, which served as a background check for mortality or growth inhibition of WCR (Maize root beetle). The concentration of dsRNA molecules in the bioassay buffer was measured using a NANODROP ^™ 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE).

Samples were tested for insect activity in a bioassay using neonatal insect larvae fed an artificial insect diet. WCR eggs were obtained from CROP CHARACTERISTICS, INC. (Farmington, MN).

Bioassays were performed in 128-well plastic trays (CD INTERNATIONAL, Pitman, NJ) specially designed for insect bioassays. Each well contained approximately 1.0 mL of artificial diet designed to support the growth of Coleopteran insects. A 60 μL aliquot of the dsRNA sample was delivered onto the surface of the diet in each well (40 μL/cm ² ) by pipette. The dsRNA sample concentration was calculated as the amount of dsRNA (ng/cm ² ) per square centimeter of surface area (1.5 cm ² ) in the well. Keep the treated trays in the fume hood until the liquid on the surface of the food evaporates or absorbs into the food.

Within a few hours of hatching, individual larvae were picked up with a moistened camel hair brush and placed on the treated food (one or two larvae per well). The infested wells of the 128-well plastic trays were then sealed with clear plastic adhesive sheets and vented to allow gas exchange. The bioassay trays were kept under controlled environmental conditions (28°C, about 40% relative humidity, 16:8 (light:dark)) for 9 days, after which the total number of insects exposed to each sample, the number of dead insects, and the number of surviving insects were recorded the weight of. Calculate the mean percent mortality and mean growth inhibition for each treatment. Growth inhibition (GI) was calculated as follows:

GI=[1–(TWIT/TNIT)/(TWIBC/TNIBC)],

where TWIT is the total weight of live insects in the treatment;

TNIT is the total number of insects in the treatment;

TWIBC is the total weight of live insects in the background check (buffer control);

TNIBC is the total number of insects in the background check (buffer control).

Statistical analysis was performed using JMP ^™ software (SAS, Cary, NC).

LC50 (Lethal Concentration) is defined as the dose at which ₅₀ % of the tested insects are killed. _GI50 (growth inhibition) is defined as the dose at which the mean growth (eg live weight) of the test insects is 50% of the mean value observed in the background check samples.

Repeated bioassays demonstrated that ingestion of specific samples caused surprising and unexpected mortality and growth inhibition of corn rootworm larvae.

Example 2: Identification of Candidate Target Genes

Insects from multiple WCR (corn root firefly beetle) developmental stages were selected for pooled transcriptome analysis to provide candidate target gene sequences controlled by RNAi technology for transgenic plant insect protection.

In one example, total RNA was isolated from approximately 0.9 g of intact first instar WCR larvae (4 to 5 days after hatch; maintained at 16°C) and analyzed using the following phenol/TRI-based The method (MOLECULAR RESEARCH CENTER, Cincinnati, OH) purification:

Place the larvae in a container containing 10 mL TRI at room temperature Homogenize in a 15 mL homogenizer until a homogeneous suspension is obtained. After 5 minutes of incubation at room temperature, the homogenate was dispensed into 1.5 mL microcentrifuge tubes (1 mL per tube), and the mixture was vigorously shaken for 15 seconds after the addition of 200 μL of chloroform. After allowing the extraction process to stand at room temperature for 10 minutes, the phases were separated by centrifugation at 12,000 xg at 4°C. Carefully transfer the upper phase (containing approximately 0.6 mL) to another sterile 1.5 mL tube and add an equal volume of room temperature isopropanol. After incubation at room temperature for 5 to 10 minutes, the mixture was centrifuged at 12,000 xg (4°C or 25°C) for 8 minutes.

The supernatant was carefully removed and discarded, then the RNA pellet was washed twice by vortexing with 75% ethanol and recovered by centrifugation at 7,500 xg (4°C or 25°C) for 5 minutes after each wash. Carefully remove the ethanol, allow the pellet to air dry for 3 to 5 min, and then dissolve in nuclease-free sterile water. RNA concentration was determined by measuring absorbance (A) at 260nm and 280nm. A typical extraction process from about 0.9 g of larvae yields over 1 mg of total RNA with an _A260 to _A280 ratio of 1.9. The RNA thus extracted was stored at -80°C until further processing.

RNA quality was determined by running aliquots through a 1% agarose gel. In an autoclaved container, use autoclaved 10x TAE buffer (Tris acetate EDTA; 1x concentration of 0.04M Tris acetate, 1 mM EDTA (sodium salt of ethylenediaminetetraacetic acid, pH 8.0) to make an agarose gel solution. Use 1x TAE as running buffer. Before use, the electrophoresis tank and comb were cleaned with RNaseAway ^™ (INVITROGEN INC., Carlsbad, CA). Take 2 μL RNA sample with 8 μL TE buffer (10 mM Tris HCl, pH 7.0; 1 mM EDTA) and 10 μL RNA sample buffer ( Cat. No. 70606; EMD4 Bioscience, Gibbstown, NJ) mix. The sample was heated at 70°C for 3 minutes, cooled to room temperature and loaded with 5 μL of sample (containing 1 μg to 2 μg RNA) per well. Commercially available RNA molecular weight markers were placed in separate wells and run simultaneously to compare molecular sizes. Run the gel at 60 volts for 2 hours.

Normalized cDNA libraries were prepared from larval total RNA using random priming by a commercial service provider (EUROFINS MWG Operon, Huntsville, AL). Normalized larval cDNA libraries were sequenced at 1/2 plate scale by GS FLX 454 Titanium ^™ series chemistry at EUROFINS MWG Operon, generating over 600,000 reads with an average read length of 348bp. 350,000 reads were assembled into more than 50,000 contigs. Both unassembled reads and contigs were converted into BLAST-capable databases using the publicly available program FORMATDB (available from NCBI).

Total RNA and normalized cDNA libraries were similarly prepared from material harvested at other WCR developmental stages. A pooled transcriptome library for screening target genes is constructed by pooling cDNA library members representing each developmental stage.

The candidate genes for RNAi targeting were hypothesized to be essential for the survival and growth of Coleopteran insects. For selected target genes, their homologues were identified in transcriptome sequence databases as described below. Templates for the production of double-stranded RNA (dsRNA) are prepared by PCR amplifying the full-length or partial sequence of the target gene.

TBLASTN searches were run against BLASTable databases containing unassembled Chrysophyllum sequence reads or assembled contigs using candidate protein-coding sequences. Significant hits (defined as better than e ⁻²⁰ for contig homology and better than e ⁻¹⁰ for unassembled sequence read homology) were confirmed against NCBI non-redundant databases using BLASTX. The results of this BLASTX search confirm that the Chromophyllum homologue candidate gene sequences identified in the TBLASTN search do indeed contain Chrysophyllia genes, or are the best hits for non-Pyrophyllum candidate gene sequences present in the Chrysophyllum sequences . In a few cases, it became evident that some of the Chrysophyllia contigs or unassembled sequence reads selected based on homology to non-Pyrophyllum candidate genes overlapped, and assembly of the contigs failed to connect these overlapping. In these cases, the sequences were assembled into longer contigs using Sequencher ^™ v4.9 (GENE CODES CORPORATION, Ann Arbor, MI).

A candidate target gene (SEQ ID NO: 1 ) encoding Chromophyll spt6 was identified as a gene that can cause death, growth inhibition, or inhibition of WCR development in coleopteran pests.

The highly conserved and multifunctional histone chaperone Spt6 protein plays a prominent role in the control of transcription in Drosophila It involves multiple steps of transcription including initiation, elongation and termination. It interacts with a variety of important factors including RNA polymerase II (RNAPII), histones and transcription factors. Spt6 can assemble nucleosomes through its interaction with nucleosomal H3/H4 tetramers and restore normal chromatin structure following RNAPII transcription.

SEQ ID NO: 1 is novel. This sequence is not available in public databases, nor in PCT International Patent Publication No. WO/2011/025860, U.S. Patent Application No. 20070124836, U.S. Patent Application No. 20090306189, U.S. Patent Application No. US20070050860, U.S. Patent Application No. 20100192265, U.S. Patent No. 7,612,194 and US Patent Application No. 2013192256. WCR spt6-1 (SEQ ID NO: 1 ) is somewhat related to a fragment of the sequence from Pediculus humanus corporis (GENBANK Accession No. XM_002424131.1). The closest homolog of the WCR SPT6-1 amino acid sequence (SEQ ID NO: 2) is the Tribolium casetanum protein, whose GENBANK accession number is XP_008196911.1 (88% similar; 79 in the homologous region %same).

The Spt6 dsRNA transgene can be combined with other dsRNA molecules to provide redundant RNAi targeting and synergistic RNAi effects. Transgenic maize events expressing dsRNA targeting spt6 can be used to prevent rhizophageal damage caused by corn rootworms. The Spt6 dsRNA transgene represents a new mode of action that can be used in conjunction with Bacillus thuringiensis insecticidal protein technology in the Insect Resistance Management gene pyramid to slow rootworm populations' response to any of these rootworm control technologies One produces resistance.

Full-length or partial clones of the sequence of the Chromophyll candidate gene (referred to herein as spt6) were used to generate PCR amplicons for dsRNA synthesis.

Example 3: Amplification of target genes to generate dsRNA

Full-length or partial clones of the sequence of the Chromophyll candidate gene (referred to herein as spt6) were used to generate PCR amplicons for dsRNA synthesis. Primers were designed to amplify the portion of the coding region of each target gene by PCR. See Table 1. The T7 phage promoter sequence (TTAATACGACTCACTATAGGGAGA; SEQ ID NO: 6) was incorporated into the 5' end of the amplified sense or antisense strand, as appropriate. See Table 1. use (Life Technologies, Grand Island, NY) extracted total DNA from WCR, then used the total DNA, using The first-strand synthesis system and the manufacturer's oligo-dT priming instructions (Life Technologies, Grand Island, NY) were used to make first-strand cDNA. The first-strand cDNA is used as a template for a PCR reaction employing reverse-positioned primers to amplify all or part of the native target gene sequence. dsRNA was also amplified from a DNA clone containing the coding region for yellow fluorescent protein (YFP) (SEQ ID NO:7; Shagin et al. (2004) Mol. Biol. Evol. 21(5):841-50) .

Table 1. Primers and primer pairs used to amplify portions of the coding region of an exemplary spt6 target gene and YFP negative control gene.

Example 4: RNAi constructs

Template preparation by PCR and dsRNA synthesis

The strategy used to provide specific templates to generate spt6 and YFP dsRNAs is shown in Figure 1. Template DNA intended for use in spt6 dsRNA synthesis was prepared by PCR using the primer pairs in Table 1, and first-strand cDNA prepared from total RNA isolated from WCR eggs, first instar larvae, or adults (as a PCR template) . For each selected spt6 and YFP target gene region, PCR amplification introduced a T7 promoter sequence at the 5' end of the amplified sense and antisense strands (the YFP segment was amplified from the YFP coding region DNA cloning). The two PCR-amplified fragments of each target gene region are then mixed in approximately equal amounts, and the mixture is used as a transcription template for the generation of dsRNA. See Figure 1. The sequences of dsRNA templates amplified with specific primer pairs are: SEQ ID NO: 3 (spt6-1 reg1), SEQ ID NO: 4 (spt6-1 v1), SEQ ID NO: 5 (spt6-1 v2) and SEQ ID NO: 5 (spt6-1 v2) ID NO: 7 (YFP). use The RNAi kit followed the manufacturer's instructions (INVITROGEN), or used In Vitro Transcription Kit Double-stranded RNA for insect bioassays was synthesized and purified following the manufacturer's instructions (New England Biolabs, Ipswich, MA). The concentration of dsRNA was measured using a NANODROP ^™ 8000 spectrophotometer (THERMO SCIENTIFIC, Wilmington, DE).

Construction of Plant Transformation Vectors

Using a combination of chemically synthesized fragments (DNA2.0, Menlo Park, CA) and standard molecular cloning methods, an entry vector containing a segment of spt6 (SEQ ID NO: 1 ) for hairpin formation was assembled. Target gene construct. Intramolecular hairpin formation of the RNA primary transcript is facilitated by arranging (within a single transcription unit) two copies of the spt6 target gene segment in opposite orientations to each other, the two segments being bounded by an adapter polynucleotide ( For example, a loop (such as SEQ ID NO:80) is separated from the ST-LS1 intron (Vancanneyt et al. (1990) Mol. Gen. Genet. 220(2):245-50)). Thus, the primary mRNA transcript contains two spt6 gene segment sequences separated by the linker sequence as large inverted repeats of each other. Use promoters (e.g., maize ubiquitin 1, U.S. Patent 5,510,474; 35S from cauliflower mosaic virus (CaMV); sugarcane baculovirus (ScBV) promoter; promoter from rice actin gene; ubiquitin promoter , pEMU, MAS, maize H3 histone promoter, ALS promoter, phaseolin gene promoter, cab, rubisco, LAT52, Zm13 and/or apg) to drive the production of primary mRNA hairpin transcripts, and use a containing Fragments of the 3' untranslated region (eg, maize peroxidase 5 gene (ZmPer5 3'UTR v2; US Patent 6,699,984), AtUbi10, AtEf1 or StPinII) are used to terminate transcription of genes expressing hairpin RNA.

in standard The above entry vector and a typical binary destination vector were used in the recombination reaction to generate an spt6 hairpin RNA expression transformation vector for Agrobacterium-mediated transformation of maize embryos.

The binary destination vector contains a plant operable promoter (for example, the sugarcane baculovirus (ScBV) promoter (Schenk et al., (1999) Plant Mol. Biol. 39:1221-30) or ZmUbi1 (US Patent 5,510,474)) Herbicide tolerance gene (aryloxyalkanoate dioxygenase; AAD-1 v3) under the regulation of (US Patent 7,838,733 (B2), and Wright et al., (2010) Proc.Natl.Acad .Sci.U.S.A.107:20240-5). The 5'UTR and linker were positioned between the 3' end of the promoter segment and the start codon of the AAD-1 coding region. Transcription of AAD-1 mRNA was terminated using a fragment containing the 3' untranslated region (ZmLip 3'UTR; US Patent No. 7,179,902) from the maize lipase gene.

With the standard use of typical binary destination vectors and entry vectors For recombination reaction, a negative control binary vector containing a gene expressing YFP protein was constructed. The binary destination vector comprises the herbicide tolerance gene (aryloxyalkanoate dioxygenase; AAD-1v3) (as above) under the expression regulation of the maize ubiquitin 1 promoter (as above). ) and a fragment comprising the 3' untranslated region (ZmLip 3'UTR; as above) from the maize lipase gene. The entry vector comprises the YFP coding region (SEQ ID NO: 14) under the expression control of the maize ubiquitin 1 promoter (as above) and a 3' untranslated region comprising the 3' untranslated region from the maize peroxidase 5 gene (as above). text) fragments.

Example 5: Screening candidate target genes

Synthetic dsRNA designed to inhibit the target gene sequences identified in Example 2, when administered to WCRs, caused death and growth inhibition in a diet-based assay.

Repeated bioassays demonstrated that ingestion of dsRNA preparations derived from spt6-1 reg1, spt6-1 v1 and spt6-1 v2 caused death and growth inhibition in western corn rootworm larvae. Table 2 shows the results of a diet-based feeding bioassay after WCR larvae were exposed to spt6-1reg1, spt6-1 v1, and spt6-1 v2 dsRNA for 9 days, together with the yellow fluorescent protein (YFP) coding region (SEQ ID NO: 7) The result obtained from the dsRNA negative control sample prepared. Table 3 shows the LC50 and GI50 results for exposure to _spt6-1 v1 and _spt6-1 v2 dsRNA.

Table 2. Results of the spt6 dsRNA diet feeding assay obtained after 9 days of feeding with western corn rootworm larvae. ANOVA analysis found significant differences in mean % mortality and mean % growth inhibition (GI). Means were separated using the Tukey-Kramer test.

*SEM = standard error of the mean Letters in parentheses indicate statistical levels. The levels not connected by the same letter are significantly different (P<0.05).

**TE = Tris HCl (1 mM) plus EDTA (0.1 mM) buffer, pH 7.2.

***YFP = yellow fluorescent protein

Table 3. Summary of oral potency (ng/cm ² ) of spt6 dsRNA against WCR larvae.

It was previously suggested that certain Chrysophyll species genes could be used for RNAi-mediated insect control. See US Patent Publication No. 2007/0124836 (which discloses 906 sequences) and US Patent No. 7,612,194 (which discloses 9,112 sequences). However, the skilled artisan determined that many of the genes proposed to be useful for RNAi-mediated insect control were not effective for the control of Chrysophyllia. It was also determined that the sequences spt6-1reg1, spt6-1 v1 and spt6-1 v2 dsRNA provided surprising and unexpected insights into Chrysophyllus compared to other genes proposed to be useful for RNAi-mediated insect control. Excellent control.

For example, in US Patent No. 7,612,194 it is proposed that annexin, beta spectrin 2, and mtRP-L4 are all effective in RNAi-mediated insect control. SEQ ID NO: 15 is the DNA sequence of Annexin Region 1 (Reg 1 ), and SEQ ID NO: 16 is the DNA sequence of Annexin Region 2 (Reg 2). SEQ ID NO: 17 is the DNA sequence of β-spectrin 2 region 1 (Reg 1), and SEQ ID NO: 18 is the DNA sequence of β-spectrin 2 region 2 (Reg 2). SEQ ID NO: 19 is the DNA sequence of mtRP-L4 region 1 (Reg1), and SEQ ID NO: 20 is the DNA sequence of mtRP-L4 region 2 (Reg 2). A YFP sequence (SEQ ID NO:7) was also used to generate dsRNA as a negative control.

Each of the aforementioned sequences was used to generate dsRNA by the method of Example 3. A strategy for providing specific templates to generate dsRNA is shown in FIG. 2 . Template DNA intended for use in dsRNA synthesis was prepared by PCR using the primer pairs in Table 4, and first strand cDNA prepared from total RNA isolated from WCR first instar larvae as a PCR template. (YFP was amplified from DNA clones.) For each selected target gene region, two separate PCR amplifications were performed. The first PCR amplification introduces a T7 promoter sequence at the 5' end of the amplified sense strand. The second reaction incorporates the T7 promoter sequence at the 5' end of the antisense strand. The two PCR-amplified fragments of each target gene region are then mixed in approximately equal amounts, and the mixture is used as a transcription template for the generation of dsRNA. See Figure 2. use RNAi kit, double-stranded RNA was synthesized and purified following the manufacturer's instructions (INVITROGEN). The concentration of dsRNA was measured using a NANODROP ^™ 8000 spectrophotometer (THERMOSCIENTIFIC, Wilmington, DE), and each dsRNA was tested by the same food-based bioassay as above. Table 4 lists the dsRNA molecules used to generate Annexin Reg 1, Annexin Reg 2, β-spectrin 2 Reg 1, β-spectrin 2 Reg 2, mtRP-L4 Reg 1, mtRP-L4 Reg 2, and YFP the sequence of the primers. Table 5 presents the results of a diet-based feeding bioassay of WCR larvae after 9 days of exposure to these dsRNA molecules. Repeated bioassays demonstrated that ingestion of these dsRNAs did not cause mortality or growth inhibition in western corn rootworm larvae beyond that seen with control samples such as TE buffer, water, or YFP protein.

Table 4. Primers used to amplify the coding region portion of the gene and

primer pair.

Table 5. Results of diet feeding assays obtained with western corn rootworm larvae after 9 days.

*TE = Tris HCl (10 mM) plus EDTA (1 mM) buffer, pH8.

**YFP = yellow fluorescent protein

Example 6: Production of insecticidal dsRNA

transgenic maize tissue

Agrobacterium-mediated transformation. Following Agrobacterium-mediated transformation, one or more insecticidal dsRNA molecules (e.g., at least one dsRNA molecule, including those targeting Transgenic maize cells, tissues and plants of the dsRNA molecule of the gene (eg SEQ ID NO: 1)). Maize transformation methods using super-binary transformation vectors or binary transformation vectors are known in the art, as described, for example, in US Patent No. 8,304,604, which is incorporated herein by reference in its entirety. Transformed tissues are selected for the ability to grow on haloxyfop-containing medium and screened for dsRNA production as appropriate. A portion of this transformed tissue culture can be provided to neonatal corn rootworm larvae to perform bioassays generally as described in Example 1.

Agrobacterium culture starts . A glycerol stock solution of Agrobacterium strain DAt13192 cells (PCT International Publication No. WO 2012/016222A2) containing the binary transformation vector described above (Example 4) was streaked on AB minimal medium plates containing appropriate antibiotics (Watson et al., (1975) J. Bacteriol. 123:255-264) and grown at 20°C for 3 days. The culture was then streaked onto YEP plates (yeast extract, 10 g/L; peptone, 10 g/L; NaCl, 5 g/L) containing the same antibiotics and incubated at 20°C for 1 day.

Agrobacterium culture . On the day of the experiment, stock solutions of inoculum medium and acetosyringone were prepared in volumes appropriate to the number of constructs in the experiment and pipetted into 250 mL disposable sterile flasks. Inoculation medium (Frame et al., (2011) Genetic Transformation Using Maize Immature Zygotic Embryos, in PlantEmbryo Culture Methods and Protocols: Methods in Molecular Biology. TAThorpe and ECYeung (eds.), Springer Science and Business Media, LLC., p. 327 -341 pages) contains: 2.2g/L MS salts, 1X ISU modified MS vitamins (Frame et al., supra), 68.4g/L sucrose, 36g/L glucose, 115mg/L L-proline, and 100mg /L inositol; pH 5.4. Add acetosyringone from a 1 M stock solution in 100% dimethyl sulfoxide to the flask containing the inoculation medium to a final concentration of 200 μM and mix the solution well.

For each construct, 1 or 2 full inoculation loops of Agrobacterium from YEP plates were suspended in 15 mL of inoculum medium/acetosyringone stock solution in 50 mL disposable sterile centrifuge tubes and measured in a spectrophotometer Optical density of the solution at 550 nm (OD ₅₅₀ ). The suspension was then diluted to an _OD550 of 0.3 to 0.4 with additional inoculation medium/acetosyringone mixture. The tube containing the Agrobacterium suspension was then placed horizontally on a platform shaker (set at room temperature, about 75 rpm) and shaken for 1 to 4 hours while embryo dissection was performed.

Ear disinfection and embryo isolation . Immature maize embryos were obtained from plants of the maize inbred line B104 (Hallauer et al., (1997) Crop Science 37:1405-1406), which were grown in the greenhouse and self- or sib-pollinated to produce ears. Ears are harvested approximately 10 to 12 days after pollination. On the day of the experiment, the ears were husked and washed by immersion in commercially available bleach (ULTRA Germicidal bleach, 6.15% sodium hypochlorite; add two drops of TWEEN 20) to a 20% solution and shake for 20 to 30 minutes to disinfect surfaces, followed by three rinses in sterile deionized water in a laminar flow hood. Immature zygotic embryos (1.8 to 2.2 mm long) were aseptically excised from each ear and distributed randomly into microcentrifuge tubes, each containing a 2.0 mL suspension of the appropriate Agrobacterium cells in liquid inoculation medium solution containing 200 μM acetosyringone and added 2 μL of 10% S233 surfactant (EVONIK INDUSTRIES; Essen, Germany). Embryos from pooled ears were used for each transformation for a given set of experiments.

Agrobacterium co-cultivation . After separation, the embryos were placed on a rocking platform for 5 minutes. The contents of the tubes were then poured onto a plate of co-cultivation medium containing 4.33 g/L MS salts, 1X ISU modified MS vitamins, 30 g/L sucrose, 700 mg/L L-proline, 3.3 mg/L dicamba (3,6-dichloro-o-anisic acid or 3,6-dichloro-2-methoxybenzoic acid) KOH solution, 100mg/L inositol, 100mg/L caseinase hydrolyzate , 15 mg/L AgNO ₃ , 200 μM acetosyringone in DMSO and 3 g/L GELZAN ^™ , pH 5.8. Remove the liquid Agrobacterium suspension with a sterile disposable pipette. The embryos are then oriented with the scutellum side up using sterile forceps with the aid of a microscope. The plate was covered, sealed with 3M ^™ MICROPORE ^™ medical tape, and placed in a 25°C incubator with continuous light of approximately 60 μmol m ⁻² s ⁻¹ photosynthetically active radiation (PAR).

Select callus and regenerate transgenic events . After the co-cultivation period, embryos were transferred to resting medium consisting of: 4.33g/L MS salts, 1X ISU modified MS vitamins, 30g/L sucrose, 700mg/L L-proline acid, 3.3mg/L dicamba KOH solution, 100mg/L inositol, 100mg/L caseinase hydrolyzate, 15mg/L AgNO ₃ , 0.5g/L MES (2-(N-morpholino)ethanesulfonic acid Monohydrate; PHYTOTECHNOLOGIES LABR.; Lenexa, KS), 250 mg/L carbenicillin and 2.3 g/L GELZAN ^™ , pH 5.8. Transfer no more than 36 embryos to each plate. Place the plate in a transparent plastic box and incubate for 7 to 10 days at 27°C under continuous light of approximately 50 μmol m ⁻² s ⁻¹ PAR. The callused embryos are then transferred (<18/plate) to selection medium I, which is composed of 100 nM R-haloxyfopic acid (0.0362 mg/L; for selection of cells containing the AAD-1 gene). callus) resting medium (as above) composition. The plate was returned to the transparent box and incubated for 7 days at 27°C with continuous light of approximately 50 μmol m ⁻² s ⁻¹ PAR. Callused embryos were then transferred (<12/plate) onto selection medium II consisting of resting medium (as above) with 500 nM R-haloxyfolic acid (0.181 mg/L) composition. The plates were returned to the clear box and incubated for 14 days at 27°C with approximately 50 μmol m ⁻² s ⁻¹ PAR in continuous light. This selection step allows further proliferation and differentiation of the transgenic callus.

Proliferating embryogenic calli were transferred (<9/plate) to pre-regeneration medium. Pre-regeneration medium contains 4.33g/L MS salts, 1X ISU modified MS vitamins, 45g/L sucrose, 350mg/L L-proline, 100mg/L inositol, 50mg/L caseinase hydrolyzate, 1.0mg/L L AgNO ₃ , 0.25g/L MES, 0.5mg/L NaOH solution of naphthaleneacetic acid, 2.5mg/L ethanol solution of abscisic acid, 1mg/L 6-benzylaminopurine, 250mg/L carbenicillin, 2.5g/L L GELZAN ^TM and 0.181 mg/L haloxyfop acid, pH 5.8. Plates were stored in transparent boxes ^and incubated for 7 days at 27 °C with continuous light at approximately 50 μmol m ⁻² s ⁻¹ PAR. The regenerating calli were then transferred (<6/plate) to regeneration medium in PHYTATRAYS ^™ (SIGMA-ALDRICH) at 28°C with 16 hours of light/8 hours of darkness (approximately 160 μmol m ⁻² s ^-1 PAR) for 14 days, or until shoots and roots emerged. Regeneration medium containing 4.33g/L MS salts, 1X ISU modified MS vitamins, 60g/L sucrose, 100mg/L inositol, 125mg/L carbenicillin, 3g/L GELLAN ^™ gel and 0.181mg/L R-pyridine Chloroxopylic acid, pH 5.8. Shootlets with primary roots are then isolated and transferred directly to elongation medium without selection. Elongation medium contained 4.33 g/L MS salts, IX ISU modified MS vitamins, 30 g/L sucrose and 3.5 g/L GELRITE ^™ at pH 5.8.

Transformed plant shoots, selected for their ability to grow on Haloxyfop-containing medium, were transplanted from PHYTATRAYS ^™ into small pots filled with growth medium (PROMIX BX; PREMIER TECH HORTICULTURE), small Pots were covered with cups or HUMI-DOMES (ARCO PLASTICS), then hardened in a CONVIRON growth chamber (day 27°C/night 24°C, 16-hour photoperiod, 50-70% RH, 200 μmol m ^-2 s ^-1 PAR) . In some cases, putative transgenic plantlets were analyzed for relative copy number of the transgene by quantitative real-time PCR assay using primers designed to detect integration of the AAD1 herbicide tolerance gene into the maize genome. In addition, qPCR assays were used to detect the presence of adapter sequences and/or target sequences in putative transformants. Selected transformed plantlets are then moved to the greenhouse for further growth and testing.

T ₀ plants were transferred and colonized in the greenhouse for bioassays and seed production . When the plants reached the V3-V4 stage, they were transplanted into IE CUSTOM BLEND (PROFILE/METRO MIX 160) soil mixture in a greenhouse (light exposure type: photosynthetic or assimilative; high light limit: 1200PAR; 16 hours day length ; 27°C during the day/24°C at night) from cultivation to flowering.

Plants to be used for insect bioassays were transplanted from small pots into TINUS ^TM 350-4 (SPENCER-LEMAIRE INDUSTRIES, Acheson, Alberta, Canada) (each One plant per event). transplanting to After approximately four days, plants are infected for bioassays.

Plants of the Ti generation are obtained by pollinating silks of _T0 transgenic plants (wherein pollen is collected from non-transgenic inbred _B104 plants or other suitable pollen donors) and planting the resulting seeds. Perform interactions when possible.

Example 7: Molecular analysis of transgenic maize tissue

Molecular analysis (eg, RT-qPCR) of maize tissue was performed on leaf and/or root samples collected from greenhouse-grown plants the day before or on the same day as the assessment of rhizophageal damage.

The results of the RT-qPCR assay of the target gene were used to verify the expression of the transgene. The presence of hairpin transcripts can also be verified using RT-qPCR assays of intervening sequences between repeat sequences in expressed RNAs (essential for the formation of dsRNA hairpin molecules). Transgenic RNA expression levels were measured relative to RNA levels of the endogenous maize gene.

A portion of the AAD1 coding region in gDNA was detected by DNA qPCR analysis to estimate the transgene insertion copy number. Samples were collected from plants grown in the environmental chamber for these analyses. Results were compared to DNA qPCR results from assays designed to detect a single copy of a portion of the native gene, and simple events (with one or two copies of the spt6 transgene) were advanced to further studies in the greenhouse.

In addition, a qPCR assay designed to detect a portion of the spectinomycin resistance gene (SpecR; contained on the binary vector plasmid outside the T-DNA) was used to determine whether the transgenic plants contained a foreign integrated plasmid backbone sequence.

RNA Transcript Expression Levels: Targeted qPCR . Callus cell events or transgenic plants were analyzed by real-time quantitative PCR (qPCR) of target sequences to determine the relative expression level of the transgene, compared to the transcript level of an internal maize gene (eg, GENBANK accession number BT069734), which The gene encodes a TIP41-like protein (ie, maize homologue of GENBANK accession number AT4G34270; tBLASTX score 74% identity). RNA was isolated using the NorgenBioTek ^™ Total RNA Isolation Kit (Norgen, Thorold, ON). Total RNA was treated with on-column DNase1 following the protocol suggested by the kit. RNA was then quantified on a NANODROP 8000 spectrophotometer (THERMO SCIENTIFIC) and concentrations were normalized to 50 ng/μL. First-strand cDNA was prepared using the High Capacity cDNA Synthesis Kit (INVITROGEN) in a reaction volume of 10 μL containing 5 μL of denatured RNA, essentially following the manufacturer’s recommended protocol. The protocol was slightly modified to include adding 10 μL of 100 μM T20VN oligonucleotide (IDT) (TTTTTTTTTTTTTTTTTTTTTVN, where V is A, C, or G, and N is A, C, G, or T; SEQ ID NO:50) to random primers 1 mL tubes of the stock mix to prepare a working stock solution of the random primer and oligo dT mix.

Following cDNA synthesis, samples were diluted 1:3 with nuclease-free water and stored at -20°C until assay time.

Real-time PCR assays for target genes and TIP41-like transcripts, respectively, were performed on a LIGHTCYCLER ^™ 480 (ROCHE DIAGNOSTICS, Indianapolis, IN) in 10 μL reaction volumes. For target gene determination, primers spt6-1 v1_62 F (SEQ ID NO:58) and spt6-1v1_360R (SEQ ID NO:56), and IDT custom oligonucleotide (Oligo) probe spt6PRB Set1 (labeled with FAM, And use Zen and Iowa Black quencher to be double quenched) to react. For TIP41-like reference gene determination, primers TIPmxF (SEQ ID NO:53) and TIPmxR (SEQ ID NO:54), and probe HXTIP (SEQ ID NO:55) labeled with HEX (hexachlorofluorescein) were used.

All assays included a negative control with no template (mixture only). To prepare a standard curve, a blank (water was added to the source well) was also included in the source plate to check for sample cross-contamination. The sequences of primers and probes are shown in Table 6. Reaction component recipes for detection of various transcripts are disclosed in Table 7 and PCR reaction conditions are summarized in Table 8. The FAM (6-carboxyfluorescein phosphoramidite) fluorescent moiety was excited at 465 nm and the fluorescence was measured at 510 nm; the corresponding values for the HEX (hexachlorofluorescein) fluorescent moiety were 533 nm and 580 nm.

Table 6. Oligonucleotide sequences used for molecular analysis of transcript levels in transgenic maize.

*TIP41-like protein.

Table 7. PCR reaction recipes for detection of transcripts.

Table 8. Thermal cycler conditions for RNA qPCR.

Using LIGHTCYCLER ^TM software v1.5, Cq values were calculated using the second derivative maximum algorithm according to the supplier's recommendation, and the data were analyzed by relative quantification. For expression analysis, expression values were calculated using the ΔΔCt method (i.e., 2-(Cq TARGET–Cq REF)), which relies on comparing the difference in Cq values between two targets, assuming that for an optimized PCR reaction, each cycle The products are all doubled, and the base value is chosen as 2.

Transcript size and integrity: Northern blot assay . In some cases, additional molecular characterization of the transgenic plants was obtained using Northern blot (Northern blot) analysis to determine the molecular size of the spt6 hairpin dsRNA in transgenic plants expressing the spt6 hairpin dsRNA.

All materials and equipment were treated with RNaseZAP (AMBION/INVITROGEN) prior to use. Tissue samples (100 mg to 500 mg) were collected into 2 mL SAFELOCK EPPENDORF tubes, disrupted in 1 mL TRIZOL (INVITROGEN) for 5 minutes with a KLECKO ^™ tissue morcellator equipped with three tungsten beads (GARCIA MANUFACTURING, Visalia, CA), and then incubated at room temperature Incubate for 10 minutes at (RT). Optionally, centrifuge the sample at 11,000 rpm for 10 min at 4 °C, then transfer the supernatant to a fresh 2 mL SAFELOCK EPPENDORF tube. After adding 200 μL of chloroform to the homogenate, mix by inverting the tube for 2 to 5 minutes, incubate at RT for 10 minutes, and centrifuge at 12,000 xg for 15 minutes at 4°C. Transfer the upper phase to a sterile 1.5 mL EPPENDORF tube, add 600 μL of 100% isopropanol, and after incubating for 10 minutes to 2 hours at RT, centrifuge at 12,000 xg for 10 minutes at 4°C to 25°C. Discard the supernatant and wash the RNA pellet twice with 1 mL of 70% ethanol, centrifuging at 7,500 x g for 10 min at 4 °C to 25 °C between washes. Discard the ethanol, briefly air-dry the pellet for 3 to 5 min, and resuspend in 50 µL of nuclease-free water.

use (THERMO-FISHER) to quantify total RNA and normalize samples to 5 μg/10 μL. Then 10 μL of glyoxal (AMBION/INVITROGEN) was added to each sample. 5 to 14 ng of DIG RNA Standard Labeling Mix (ROCHE APPLIED SCIENCE, Indianapolis, IN) was dispensed and added to an equal volume of glyoxal. Samples and labeled RNA were denatured at 50°C for 45 minutes and then stored on ice until loading onto 1.25% SEAKEMGOLD agarose (LONZA, Allendale, NJ) in NORTHERNMAX 10X glyoxal running buffer (AMBION/INVITROGEN) on the gel. RNA was isolated by electrophoresis at 65V/30mA for 2 hours 15 minutes.

After electrophoresis, the gel was rinsed in 2X SSC for 5 minutes and then imaged on a GEL DOC workstation (BIORAD, Hercules, CA), followed by overnight at RT to allow passive transfer of RNA to a nylon membrane (MILLIPORE) using 10X SSC was used as transfer buffer (20X SSC consisting of 3M sodium chloride and 300M trisodium citrate, pH 7.0). After transfer, the membrane was rinsed in 2X SSC for 5 minutes, the RNA was crosslinked to the membrane using UV light (AGILENT/STRATAGENE), and the membrane was allowed to dry at room temperature for a maximum of 2 days.

Membranes were prehybridized in ULTRAHYB ^™ buffer (AMBION/INVITROGEN) for 1 to 2 hours. Probes consisted of PCR amplification products containing sequences of interest (eg, antisense portions of SEQ ID NO: 3-5, as appropriate), labeled with digoxigenin by means of the ROCHE APPLIED SCIENCE DIG program. Hybridize overnight at 60°C in the recommended buffer in the hybridization tube. After hybridization, the blots were washed in DIG, packaged, exposed to film for 1 to 30 minutes, and then developed, all by the method recommended by the supplier of the DIG kit.

Determine transgene copy number . Maize leaves, approximately equivalent to 2 leaf punches, were collected in 96-well collection plates (QIAGEN). Tissue disruption was performed in BIOSPRINT96AP1 lysis buffer (supplied with BIOSPRINT96PLANT KIT; QIAGEN) with a KLECKO ^™ tissue disruptor (GARCIAMANUFACTURING, Visalia, CA) equipped with one stainless steel bead. After tissue maceration, gDNA was isolated in a high-throughput format using the BIOSPRINT96PLANT KIT and the BIOSPRINT96 extraction robot. Before setting up the qPCR reaction, dilute gDNA with 1:3 DNA:water.

qPCR analysis . by using Systematic real-time PCR to perform transgene detection with the aid of hydrolysis probe assays. use Probe Design Software 2.0 is designed to be used in hydrolysis probe assays for detection of target genes (e.g., spt6), linker sequences (e.g., loop, SEQ ID NO: 80) and/or for detection of SpecR genes (i.e., loaded in two Spectinomycin resistance gene on metavector plasmid; SEQ ID NO:62; SPC1 oligonucleotide in Table 9) part of the oligonucleotide). In addition, the region to be used in the hydrolysis probe assay to detect the AAD-1 herbicide tolerance gene (SEQ ID NO: 63; GAAD1 oligonucleotide in Table 9) was designed using the PRIMER EXPRESS software (APPLIEDBIOSYSTEMS) Segment of oligonucleotides. Table 9 shows the sequences of primers and probes. With the reagent of endogenous maize chromosomal gene (invertase (SEQ ID NO:64; GENBANK accession number U16123; being referred to as IVR1 in this text), said gene is used as internal reference sequence to ensure that in each determination gDNA was present in both. For amplification, a 1x final concentration of Probe master mix (ROCHE APPLIED SCIENCE) containing 0.4 μM of each primer and 0.2 μM of each probe (Table 10). A two-step amplification reaction was performed as outlined in Table 11. Fluorophore activation and emission for FAM-labeled probes and HEX-labeled probes was as described above; the CY5 conjugate has an excitation maximum at 650 nm and a fluorescence maximum at 670 nm.

Using the fit point algorithm ( Software release version 1.5) and relative quantification module (based on the ΔΔCt method), the Cp score (point at which the fluorescent signal crosses the background threshold) was determined from real-time PCR data. Data were processed as previously described (above; RNAqPCR).

Table 9. Sequences of primers and probes (with fluorescent conjugates) for determination of gene copy number and detection of binary vector plasmid backbone.

*CY5 = Cyanine-5

Table 10. Reaction components for analysis of gene copy number and detection of plasmid backbone.

*NA = not applicable

**ND = not determined

Table 11. Thermal cycler conditions for qPCR.

Example 8: Bioassays of Transgenic Maize

Insect bioassays . The biological activity of the dsRNA of the subject invention produced in plant cells was confirmed by bioassay methods. See, eg, Baum et al., (2007) Nat. Biotechnol. 25(11):1322-1326. The skilled person is able to demonstrate efficacy, for example, by feeding the target insect under a controlled feeding environment various plant tissues or tissue pieces derived from the plant producing the insecticidal dsRNA. Alternatively, extracts were prepared from various plant tissues derived from plants producing insecticidal dsRNAs, and the extracted nucleic acids were distributed over artificial diets for bioassays as previously described herein. The results of such feeding assays are compared to similarly performed bioassays using appropriate control tissue from host plants that do not produce the insecticidal dsRNA, or other control samples. The growth and survival of the target insects on the test diet was reduced compared to the control group.

Insect bioassays using transgenic maize events . Two western corn rootworm larvae (1 to 3 days old) hatched from the washed eggs were selected and placed in each well of the bioassay tray. The wells were then covered with "PULL N'PEEL" covers (BIO-CV-16, BIO-SERV) and placed in a 28°C incubator with an 18h/6h light/dark cycle middle. Nine days after the initial infestation, larval mortality was assessed as a percentage of dead insects out of the total number of insects for each treatment. Insect samples were frozen at -20°C for two days, then insect larvae from each treatment were pooled and weighed. Percent growth inhibition was calculated as the mean of the mean weight of the experimental treatment divided by the mean weight of the two control well treatments. Data are expressed as percent growth inhibition (of the negative control). The mean weight over the control mean weight was normalized to zero.

Insect bioassays in the greenhouse . Soil with western corn rootworm (WCR, corn rootworm) eggs was received from CROP CHARACTERISTICS (Farmington, MN). Incubate WCR eggs at 28°C for 10 to 11 days. The soil was washed off the eggs, and the eggs were placed in a 0.15% agar solution at a concentration adjusted to approximately 75 to 100 eggs per 0.25 mL aliquot. An aliquot of the egg suspension was added to a Petri dish to set up the incubation plate for monitoring the rate of hatching.

Infestation with 150 to 200 WCR eggs Soil around maize plants growing in. Insects were allowed to feed for 2 weeks, after which time a "root rating" was given for each plant. Grading was performed using the Nodal Damage Scale, generally according to Oleson et al. (2005) J. Econ. Entomol. 98:1-8. Plants that passed the bioassay showing reduced damage were transplanted into 5 gallon pots for seed production. Treat transplants with insecticide to prevent further rootworm damage and release of insects into the greenhouse. Plants were hand pollinated to produce seeds. Seeds produced by these plants were saved for evaluation of the T1 and subsequent generations _of plants.

Transgenic negative control plants were generated by transformation with a vector containing a gene designed to produce yellow fluorescent protein (YFP). Non-transformed negative control plants are grown from seeds of the parent maize variety from which the transgenic plants were derived. Bioassays were performed in which a negative control was included in each set of plant material.

Example 9: Transgenic corn comprising sequences from coleopteran pests

From 10 to 20 transgenic _T0 maize plants were generated as described in Example 6. An additional 10 to 20 independent lines _of T1 maize expressing the hairpin dsRNA of the RNAi construct were obtained for corn rootworm challenge. The hairpin dsRNA comprises a portion of SEQ ID NO:1. Additional hairpin dsRNAs are derived, for example, from coleopteran pest sequences such as Caf1-180 (US Patent Application Publication No. 2012/0174258), VatpaseC (US Patent Application Publication No. 2012/0174259), Rho1 (US Patent Application Publication No. 2012/0174260 ), VatpaseH (US Patent Application Publication No. 2012/0198586), PPI-87B (US Patent Application Publication No. 2013/0091600), RPA70 (US Patent Application Publication No. ), ROP (US Patent Application No. 14/577,811), RNA polymerase II140 (US Patent Application No. 14/577,854), Dre4 (US Patent Application No. 14/705,807), ncm (US Patent Application No. 62/095487), COPIα (U.S. Patent Application No. 62/063,199), COPIβ (U.S. Patent Application No. 62/063,203), COPIγ (U.S. Patent Application No. 62/063,192) or COPIδ (U.S. Patent Application No. 62/063,216), RNA polymerase I1 (U.S. Patent Application No. Application No. 62/133,214), RNA Polymerase II-215 (US Patent Application No. 62/133,202), RNA Polymerase 33 (US Patent Application No. 62/133,210), and spt5 (US Patent Application No. 62/168613). These are confirmed by RT-PCR or other molecular analysis methods.

Total RNA preparations from selected independent T1 lines _were optionally used for RT-PCR with primers designed to bind in the adapter of the hairpin expression cassette in each RNAi construct. In addition, specific primers for each target gene in the RNAi construct are optionally used to amplify the preprocessed mRNA required for siRNA production in plants and to confirm that the preprocessed mRNA is produced. Amplification of the expected bands for each target gene confirmed expression of the hairpin RNA in each transgenic maize plant. Hairpin processing of the dsRNA of the target gene to siRNA is then optionally confirmed in independent transgenic lines using Northern blot hybridization.

Additionally, RNAi molecules with mismatched sequences with more than 80% sequence identity to the target gene affected corn rootworms in a manner similar to that seen when using RNAi molecules with 100% sequence identity to the target gene. Pairing of mismatched sequences with native sequences forms hairpin dsRNAs in the same RNAi construct, thereby delivering plant-processed siRNAs capable of affecting growth, development and viability of feeding coleopteran pests.

Delivery of dsRNA, siRNA, or miRNA corresponding to a target gene in plants, which is subsequently ingested by coleopteran pests through feeding, results in the downregulation of the target gene in the coleopteran pest due to RNA-mediated gene silencing. The growth and/or development of coleopteran pests is affected when the target gene plays an important role in one or more stages of development, and the effects of WCR, NCR, SCR, MCR, Cucumber-rooted beetle, South American leaf beetle, Cucumber In the case of at least one of D.u. undecimpunctata Mannerheim and D.u. undecimpunctata Mannerheim, the coleopteran pest is unable to successfully infect, feed and/or develop, or the coleopteran pest is killed. Coleopteran pests are then controlled through selection of target genes and successful application of RNAi.

Phenotypic comparison of transgenic RNAi lines and non-transformed maize. The target coleopteran pest genes or sequences chosen to create the hairpin dsRNA had no similarity to any known plant gene sequence. Therefore, the production or activation of (systemic) RNAi by constructs targeting these coleopteran pest genes or sequences is not expected to have any deleterious effects on the transgenic plants. However, the developmental and morphological characteristics of the transgenic lines were compared to non-transformed plants, as well as those transgenic lines transformed with an "empty" vector without the hairpin expressed gene. Roots, shoots, leaves, and reproductive characteristics of the plants were compared. There were no observable differences in root length and growth pattern between transgenic and non-transformed plants. Bud characteristics of the plants were recorded, such as height, leaf number and size, flowering time, flower size and appearance. In general, there were no observable morphological differences between transgenic lines and those lines that did not express the target iRNA molecule when grown in vitro and in greenhouse soil.

Example 10: Transgenic Maize Comprising Coleopteran Pest Sequences and Additional RNAi Constructs

Transgenic maize plants containing in their genome heterologous coding sequences transcribed into iRNA molecules targeting organisms other than coleopteran pests, such transgenic maize plants via the Agrobacterium or WHISKERS ^™ method (See Petolino and Arnold, (2009) Methods Mol.Biol.526:59-67) Perform secondary transformation to produce one or more insecticidal dsRNA molecules (for example, at least one dsRNA molecule, including targeting dsRNA molecule of the gene of IDNO:1). Plant transformation plasmid vectors were prepared substantially as described in Example 4 and delivered via Agrobacterium- or WHISKERS ^™ -mediated transformation methods into maize suspension cells or immature maize embryos obtained from transgenic Hi II or B104 maize plants, Maize plants contain in their genome heterologous coding sequences that are transcribed into iRNA molecules targeting organisms other than coleopteran pests.

Example 11: Transgenic Maize Comprising RNAi Constructs and Additional Coleopteran Pest Control Sequences

The transgenic maize plant comprises in its genome a heterologous coding sequence transcribed into an iRNA molecule targeting a coleopteran pest organism (e.g., at least one dsRNA molecule, including a dsRNA molecule targeting a gene comprising SEQ ID NO: 1 ), such transgenic maize plants are subjected to secondary transformation via Agrobacterium or WHISKERS ^TM methods (see Petolino and Arnold, (2009) Methods Mol. Biol. 526:59-67) to produce one or more insecticidal proteins Molecules such as Cry3, Cry34, Cry35, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C pesticidal proteins. Plant transformation plasmid vectors were prepared substantially as described in Example 4 and delivered via Agrobacterium- or WHISKERS ^™ -mediated transformation methods into maize suspension cells or immature maize embryos obtained from transgenic B104 maize plants in Its genome contains heterologous coding sequences that are transcribed into iRNA molecules targeting coleopteran pest organisms. Double transformed plants producing iRNA molecules and insecticidal proteins for controlling coleopteran pests are obtained.

Example 12: spt6 dsRNA in insect management

The Spt6 dsRNA transgene was combined with other dsRNA molecules in transgenic plants to provide redundant RNAi targeting and synergistic RNAi effects. Transgenic plants expressing dsRNA targeting spt6, including for example but not limited to maize, can be used to prevent feeding damage by coleopteran insects. The Spt6 dsRNA transgene was also combined with B. thuringiensis insecticidal protein technology in plants to represent a new mode of action in the accumulation of insect resistance regulatory genes. When combined with other dsRNA molecules and/or insecticidal proteins targeting insect pests in transgenic plants, a synergistic insecticidal effect was observed that also slowed the growth of resistant insect populations.

sequence listing

<110> Dow AgroSciences, LLC

FIME

Narva, Kenneth E

Worden, Sarah

Frey, Meghan

Rangasamy, Murugesan

Gandra, Premchand

Lo, Wendy

Fishilevich, Elane

Vilcinskas, Andreas

Knorr, Eileen

<120> SPT6 nucleic acid molecules for controlling insect pests

<130> 2971-P12796.1US (77541-US-NP)

<150> US 62/168,606

<151> 2015-05-29

<160> 84

<170> PatentIn Version 3.5

<210> 1

<211> 5948

<212>DNA

<213> Corn root firefly beetle

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ataatttt 5948

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<213> Corn root firefly beetle

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Glu Leu Asp His Arg Asp Arg Lys Lys Ala Gln Lys Ala Lys Val Val

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Asp Ser Ser Asp Glu Asp Asp Glu Asp Asp Asp Glu Arg Leu Arg Glu

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Glu Leu Lys Asp Leu Ile Asp Asp Asn Pro Ile Glu Glu Ser Asp Ala

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Glu Ser Asp Ala Ser Gly Arg Glu Lys Arg Lys Lys Ser Asp Asp Glu

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Asp Leu Asp Asp Arg Leu Glu Asp Glu Asp Tyr Asp Leu Leu Glu Glu Glu

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Asn Leu Gly Val Lys Val Glu Arg Arg Lys Phe Lys Arg Leu Arg Arg

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Phe Glu Asp Glu Glu Ser Glu Gly Glu Glu Glu His Asp Pro Glu Gln

115 120 125

Asp Arg Glu Gln Ile Ala Met Asp Ile Phe Ser Asp Asp Asp Asp Glu

130 135 140

Arg Arg Ser Glu Arg Ser His Arg Pro Ala Val Glu Gln Glu Thr Tyr

145 150 155 160

Gly Val Gly Glu Glu Glu Glu Glu Gly Glu Tyr Ser Asp Ala Asp Asp

165 170 175

Phe Ile Val Asp Asp Asp Asp Gly Arg Pro Ile Ala Glu Lys Lys Lys Lys Lys

180 185 190

Lys Lys Pro Ile Phe Thr Asp Ala Ala Leu Gln Glu Ala Gln Glu Thr

195 200 205

Phe Gly Val Asp Phe Asp Tyr Asp Glu Phe Ser Lys Tyr Asp Glu Asp

210 215 220

Asp Tyr Glu Asp Glu Glu Glu Glu Asp Asp Glu Tyr Glu Glu Asp Asp

225 230 235 240

Val Glu Lys Arg Lys Arg Pro Lys Lys Thr Ser Lys Lys Lys Pro Thr

245 250 255

Lys Lys Ser Ile Phe Glu Val Tyr Glu Pro Ser Glu Leu Lys Arg Gly

260 265 270

Phe Phe Thr Asp Leu Asp Asn Glu Ile Arg Asn Thr Asp Ile Pro Glu

275 280 285

Arg Met Gln Leu Arg Asp Val Pro Ile Thr Ala Val Pro Asp Asp Ser

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Thr Glu Leu Asp Asp Glu Ala Glu Trp Ile Tyr Arg Gln Ala Phe Cys

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Asn Arg Thr Val Ser Asn Val Asp Ser His Leu Ser Ser Glu Ala Arg

325 330 335

Glu Lys Leu Lys Lys Thr His His Ala Ile Gly Lys Ile Arg Lys Ala

340 345 350

Leu Asp Phe Ile Arg Asn Gln Gln Leu Glu Val Pro Phe Ile Ala Phe

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Tyr Arg Lys Glu Tyr Val Gln Pro Glu Leu Asn Ile Asn Asp Leu Trp

370 375 380

Lys Val Tyr Lys Tyr Asp Ala Lys Trp Cys Gln Leu Lys Thr Arg Lys

385 390 395 400

Glu Asn Leu Leu Lys Leu Phe Glu Lys Met Arg Ser Tyr Gln Thr Asp

405 410 415

His Ile Met Lys Asp Pro Asp Ala Pro Ile Pro Asp Asn Leu Arg Ile

420 425 430

Met Thr Glu Ser Asp Ile Glu Arg Leu Lys Asn Val Gln Thr Ala Glu

435 440 445

Glu Leu Asn Asp Val His Asn His Phe Ile Leu Tyr Tyr Ala Ala Asp

450 455 460

Leu Pro Ala Met His Ala Ala Trp Arg Val Lys Glu Arg Glu Arg Arg

465 470 475 480

Arg Gln Glu Arg Lys Glu Ala Arg Leu Lys Leu Ile Ala Glu Ser Glu

485 490 495

Glu Gly Ala Glu Ile Pro Glu Glu Pro Glu Glu Ile Asp Asp Asp Glu

500 505 510

Pro Glu Ala Glu Thr Leu Lys Tyr Ala Asn Arg Ser Gly Ser Tyr Ala

515 520 525

Leu Cys Asn Lys Gly Gly Leu Gly Pro Leu Ala Lys Lys Phe Gly Leu

530 535 540

Thr Pro Glu Glu Phe Ala Glu Asn Leu Arg Asp Asn Tyr Gln Arg His

545 550 555 560

Glu Val Asp Gln Glu His Ile Glu Pro Val Glu Val Ala Lys Glu Phe

565 570 575

Val Ser Pro Lys Phe Pro Thr Val Glu Glu Val Leu Gln Ala Thr Lys

580 585 590

His Met Val Ala Leu Gln Ile Ala Arg Glu Pro Leu Val Arg Lys Cys

595 600 605

Val Arg Glu Ile Phe Phe Glu Arg Ala Arg Leu Asn Val Tyr Pro Thr

610 615 620

Lys Lys Gly Val Lys Val Ile Asp Glu Ala His Asn Cys Tyr Ser Met

625 630 635 640

Lys Tyr Val Lys Asn Lys Pro Val Arg Asp Leu Ala Gly Asp Gln Phe

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Leu Lys Leu Cys Leu Ala Glu Glu Glu Asn Leu Leu Thr Ile Thr Ile

660 665 670

Asn Asp His Ile Gln Gly Asn Thr Thr Asn Asn Asn Tyr Ile Asp Glu Val

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Lys Gln Leu Tyr Ile Lys Asp Glu Phe Ser Lys His Val Gln Asp Trp

690 695 700

Asn Ala Leu Arg Met Glu Ser Val Glu Arg Ala Leu Thr Lys Ser Val

705 710 715 720

Leu Pro Asp Leu Arg Ser Glu Leu Lys Arg Thr Leu Leu Thr Glu Ala

725 730 735

Lys Glu Phe Val Leu Lys Ala Cys Cys Arg Lys Leu Tyr Asn Trp Ile

740 745 750

Lys Ile Ala Pro Tyr Ala Ile Thr Phe Pro Asp Glu Asp Glu Asp Asp

755 760 765

Trp Asp Thr Ser Lys Gly Val Arg Thr Met Gly Val Ala Tyr Val Pro

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Glu His Thr Val Ser Ala Phe Thr Cys Ile Ser Ala Pro Asp Gly Asp

785 790 795 800

Ile Thr Asp Tyr Leu Arg Leu Pro Asn Ile Leu Lys Arg Lys Asn Ser

805 810 815

Phe Arg Thr Glu Glu Lys Leu Met Lys Glu Ala Asp Leu Gln Ala Leu

820 825 830

Lys Asn Phe Ile Phe Leu Lys Lys Pro His Val Ile Ala Val Gly Gly

835 840 845

Glu Ser Arg Glu Ala Leu Met Ile Ala Asp Asp Ile Arg Gly Val Ile

850 855 860

Ser Glu Leu Ile Glu Ser Asp Gln Phe Pro Gln Ile Arg Val Glu Ile

865 870 875 880

Ile Asp Asn Glu Leu Ala Lys Val Tyr Ala Asn Ser Ile Lys Gly Ser

885 890 895

Thr Asp Phe Arg Asp Tyr Pro Glu Leu Leu Arg Gln Ala Ile Ser Leu

900 905 910

Ala Arg Arg Met Gln Asp Pro Leu Val Glu Phe Ser Gln Leu Cys Asn

915 920 925

Ser Asp Glu Glu Ile Leu Ser Leu Arg Phe His Pro Leu Gln Glu Gln

930 935 940

Val Gln Lys Glu Glu Leu Leu Glu Ala Leu Cys Leu Glu Phe Val Asn

945 950 955 960

Arg Thr Asn Glu Val Gly Val Asp Ile Asn Leu Ala Val Gln Gln Ile

965 970 975

His Lys Ser Ser Leu Val Gln Phe Ile Cys Gly Leu Gly Pro Arg Lys

980 985 990

Gly Gln Ala Leu Leu Lys Val Leu Lys Gln Thr Asn Gln Arg Leu Glu

995 1000 1005

Asn Arg Thr Gln Leu Val Thr Phe Cys His Met Gly Pro Lys Val

1010 1015 1020

Phe Ile Asn Cys Ser Gly Phe Ile Lys Ile Asp Thr Asn Ser Leu

1025 1030 1035

Gly Asp Ser Thr Glu Ala Tyr Val Glu Ile Leu Asp Gly Ser Arg

1040 1045 1050

Val His Pro Glu Thr Tyr Glu Trp Ala Arg Lys Met Ala Val Asp

1055 1060 1065

Ala Leu Glu Tyr Asp Asp Asp Glu Gly Ala Asn Pro Ala Gly Ala

1070 1075 1080

Leu Glu Glu Ile Leu Glu Ala Pro Glu Arg Leu Lys Asp Leu Asp

1085 1090 1095

Leu Asp Ala Phe Ala Glu Glu Leu Glu Arg Gln Gly Phe Gly Asn

1100 1105 1110

Lys Ser Ile Thr Leu Tyr Asp Ile Arg Ala Glu Leu Asn Ser Arg

1115 1120 1125

Tyr Lys Asp Leu Arg Gln Pro Phe Arg Ser Ala Asn Pro Glu Glu

1130 1135 1140

Leu Phe Asp Met Leu Thr Lys Glu Thr Pro Glu Thr Phe Tyr Ile

1145 1150 1155

Gly Lys Met Val Thr Ser Thr Val Phe Gly Ile Ala Arg Arg Lys

1160 1165 1170

Pro Lys Ser Asp Gln Leu Asp Gln Ala Asn Pro Val Arg Asn Asp

1175 1180 1185

Glu Thr Gly Leu Trp Gln Cys Pro Phe Cys Leu Lys Asn Asp Phe

1190 1195 1200

Pro Glu Leu Ser Asp Val Trp Asn His Phe Asp Ala Gly Ala Cys

1205 1210 1215

Pro Gly Gln Ala Thr Gly Val Lys Leu Arg Leu Asp Asn Gly Ile

1220 1225 1230

Leu Gly Tyr Ile Tyr Ile Lys Asn Ile Ser Asp Lys Pro Val Ala

1235 1240 1245

Asn Pro Glu Glu Arg Val Gly Ile Gly Gln Leu Ile His Cys Arg

1250 1255 1260

Ile Ile Lys Ile Asp Val Glu Lys Phe Ser Val Asp Cys Thr Ser

1265 1270 1275

Lys Ser Ser Asp Leu Ala Asp Lys Asn His Glu Trp Arg Pro Gln

1280 1285 1290

Arg Asp Pro His Tyr Asp Gln Glu Arg Glu Asp Lys Asp Asn Arg

1295 1300 1305

Leu Glu Ala Glu Lys Lys Lys Glu Lys Gln Arg Met Thr Tyr Ile

1310 1315 1320

Lys Arg Val Ile Val His Pro Ala Phe His Asn Ile Ser Tyr Ala

1325 1330 1335

Glu Ala Glu Lys Cys Met Val Asn Met Asp Gln Gly Glu Val Ile

1340 1345 1350

Ile Arg Pro Ser Ser Lys Gly Ala Asp His Leu Thr Ile Thr Trp

1355 1360 1365

Lys Val Thr Asp Gly Ile Tyr Gln His Ile Asp Ile Lys Glu Gln

1370 1375 1380

Gly Lys Val Asn Ala Phe Ser Leu Gly Lys Ser Leu Trp Ile Gly

1385 1390 1395

Asn Glu Glu Phe Glu Asp Leu Asp Glu Ile Ile Ala Arg His Val

1400 1405 1410

Thr Pro Met Ala Ala His Ala Arg Asp Leu Leu Tyr Phe Arg Tyr

1415 1420 1425

Tyr Lys Asp Phe Gln Gly Gly His Lys Asp Lys Ala Glu Glu Tyr

1430 1435 1440

Leu Lys Asp Glu Lys Lys Lys Asn Ala Ser Lys Ile His Tyr Val

1445 1450 1455

Val Ser Ala Ala Lys Asn Ile Pro Gly Lys Phe Leu Leu Ser Tyr

1460 1465 1470

Leu Pro Arg Asn Lys Val Arg His Glu Tyr Val Thr Val Thr Pro

1475 1480 1485

Glu Gly Phe Arg Phe Arg Gln Gln Met Phe Asp Ser Val Ser Ser

1490 1495 1500

Leu Phe Lys Trp Phe Lys Glu His Phe Arg Glu Pro Pro Pro Gly

1505 1510 1515

Gly Ala Thr Pro Gly Ser Thr Pro Arg Met Ala Ser Ser Arg Thr

1520 1525 1530

Gly Tyr Gly Ser Ala Thr Pro Ala Tyr Ser Met Asn Asn Glu Ala

1535 1540 1545

Ile Gln Arg Val Ala Gln Asn Leu Pro Ser His Val Val Gln Ala

1550 1555 1560

Leu Ser Ala Ala Thr Asn Gln Thr Pro His Tyr Pro His Thr Pro

1565 1570 1575

Gly Tyr Gly Gly Asn Tyr Ile Asn Thr Pro Tyr Thr Pro Ser Gly

1580 1585 1590

Gln Thr Pro Tyr Met Thr Pro Tyr Ala Thr Pro His Thr Gln Gln

1595 1600 1605

Thr Pro Arg Tyr Gly His Gln Thr Pro Ser Gln His Met Ala Ser

1610 1615 1620

Ser Ala Pro Gln Gly Leu Asn Asn Pro Phe Leu His Pro Gly Ala

1625 1630 1635

Val Thr Pro Ser Gln Arg Thr Pro Ile Tyr Arg Asn His Pro Ala

1640 1645 1650

Gln Ser Pro Val Met Leu Pro Thr Ser Pro Val Pro Ser Pro Gly

1655 1660 1665

Ser Gln Ser Ser Tyr Ser Ser His Leu Ser His Asn Gln Arg Ser

1670 1675 1680

Gly Ser Tyr Ala Glu Ser Leu Arg Phe Gln Pro Pro Glu Ser Pro

1685 1690 1695

Arg Ser Ser Val Ser Asn Arg Ser Phe Gln Thr Asp Arg Tyr Gly

1700 1705 1710

Gly Asp Arg Tyr Gly Lys Gly Gly Ser His Arg Tyr Gly Gly Ser

1715 1720 1725

Ser Asn Glu Asp Arg Tyr Gly Lys Gly Gly Gly Gly Asn Glu Asn

1730 1735 1740

Thr Asp Trp Gln Lys Ala Ala Glu Ala Trp Ala Arg Ser Arg Ser

1745 1750 1755

Thr Pro Arg Ser Asp Gly Arg Asn Thr Pro Arg Ser Val Gly Gln

1760 1765 1770

Arg Thr Pro Arg Tyr Asp Asn Asp Ala Glu Arg Ser Arg Met Lys

1775 1780 1785

His Leu Ser Lys Ser Pro Arg Ser Val Arg Ser Thr Pro Arg Thr

1790 1795 1800

Asn Thr Ser Pro His Ser Met Ser Leu Gly Asp Ala Thr Pro Leu

1805 1810 1815

Tyr Asp Glu Ser Ile

1820

<210> 3

<211> 499

<212>DNA

<213> Corn root firefly beetle

<400> 3

cgccttacac tccaagtggt caaactccat acatgactcc gtacgctaca ccccatacgc 60

agcaaactcc ccgctatggt catcaaacac cttcccaaca catggcgagc tcagcaccac 120

aaggtctcaa caatcccttt ttacatcctg gcgcggtgac tccctcccaa cgaactccta 180

tttatcgcaa ccatcctgca caatctccag taatgcttcc tacaagccct gtaccaagtc 240

caggttcccca gagttcatac agtagtcatt taagtcataa tcagcgaagt ggaagttatg 300

ctgaatcctt aagattccaa cctcccgaat cgccgagaag ctcagtgagt aatagaagct 360

ttcaaactga tagatacggc ggtgataggt atggtaaagg aggaagtcat agatatggtg 420

gaagctcaaa cgaagataga tatggtaagg gaggaggagg aaatgaaaat acggattggc 480

agaaagctgc agaagcatg 499

<210> 4

<211> 145

<212>DNA

<213> Corn root firefly beetle

<400> 4

gactccgtac gctacaccccc atacgcagca aactccccgc tatggtcatc aaacaccttc 60

ccaacacatg gcgagctcag caccacaagg tctcaacaat ccctttttac atcctggcgc 120

ggtgactccc tcccaacgaa ctcct 145

<210> 5

<211> 108

<212>DNA

<213> Corn root firefly beetle

<400> 5

agagttcata cagtagtcat ttaagtcata atcagcgaag tggaagttat gctgaatcct 60

taagattcca acctcccgaa tcgccgagaa gctcagtgag taatagaa 108

<210> 6

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> T7 phage promoter

<400> 6

ttaatacgac tcactatagg gaga 24

<210> 7

<211> 503

<212>DNA

<213> Artificial sequence

<220>

<223> Partial YFP coding sequence

<400> 7

caccatgggc tccagcggcg ccctgctgtt ccacggcaag atcccctacg tggtggagat 60

ggagggcaat gtggatggcc acaccttcag catccgcggc aagggctacg gcgatgccag 120

cgtgggcaag gtggatgccc agttcatctg caccaccggc gatgtgcccg tgccctggag 180

caccctggtg accaccctga cctacggcgc ccagtgcttc gccaagtacg gccccgagct 240

gaaggatttc tacaagagct gcatgcccga tggctacgtg caggagcgca ccatcacctt 300

cgagggcgat ggcaatttca agacccgcgc cgaggtgacc ttcgagaatg gcagcgtgta 360

caatcgcgtg aagctgaatg gccagggctt caagaaggat ggccacgtgc tgggcaagaa 420

tctggagttc aatttcaccc cccactgcct gtacatctgg ggcgatcagg ccaatcacgg 480

cctgaagagc gccttcaaga tct 503

<210> 8

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6-1_For

<400> 8

ttaatacgac tcactatagg gagacgcctt acactccaag tggtcaaac 49

<210> 9

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6-1_Rev

<400> 9

ttaatacgac tcactatagg gagacatgct tctgcagctt tctgccaatc 50

<210> 10

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6_v1_For

<400> 10

ttaatacgac tcactatagg gagagactcc gtacgctaca ccccatac 48

<210> 11

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6-1_v1_Rev

<400> 11

ttaatacgac tcactatagg gagaaggagt tcgttgggag ggagtcac 48

<210> 12

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6-1_v2_For

<400> 12

ttaatacgac tcactatagg gagaagagtt catacagtag tcatttaagt c 51

<210> 13

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Dvv-spt6-1_v2_Rev

<400> 13

ttaatacgac tcactatagg gagattctat tactcactga gcttctcg 48

<210> 14

<211> 705

<212>DNA

<213> Artificial sequence

<220>

<223> Synthetic YFP coding sequence

<400> 14

atgtcatctg gagcacttct ctttcatggg aagattcctt acgttgtgga gatggaaggg 60

aatgttgatg gccacaccctt tagcatacgt gggaaaggct acggagatgc ctcagtggga 120

aaggttgatg cacagttcat ctgcacaact ggtgatgttc ctgtgccttg gagcacactt 180

gtcaccactc tcacctatgg agcacagtgc tttgccaagt atggtccaga gttgaaggac 240

ttctacaagt cctgtatgcc agatggctat gtgcaagagc gcacaatcac ctttgaagga 300

gatggcaact tcaagactag ggctgaagtc acctttgaga atgggtctgt ctacaatagg 360

gtcaaactca atggtcaagg cttcaagaaa gatggtcatg tgttgggaaa gaacttggag 420

ttcaacttca ctccccactg cctctacatc tggggtgacc aagccaacca cggtctcaag 480

tcagccttca agatctgtca tgagattact ggcagcaaag gcgacttcat agtggctgac 540

cacacccaga tgaacactcc cattggtgga ggtccagttc atgttccaga gtatcatcac 600

atgtcttacc atgtgaaact ttccaaagat gtgacagacc acagagacaa catgtccttg 660

aaagaaactg tcagagctgt tgactgtcgc aagacctacc tttga 705

<210> 15

<211> 218

<212>DNA

<213> Corn root firefly beetle

<400> 15

tagctctgat gacagagccc atcgagtttc aagccaaaca gttgcataaa gctatcagcg 60

gattgggaac tgatgaaagt acaatmgtmg aaattttaag tgtmcacaac aacgatgaga 120

ttataagaat ttcccaggcc tatgaaggat tgtaccaacg mtcattggaa tctgatatca 180

aaggagatac ctcaggaaca ttaaaaaaga atttatag 218

<210> 16

<211> 424

<212>DNA

<213> Corn root firefly beetle

<220>

<221> misc_feature

<222> (393)..(395)

<223> n is a, c, g or t

<400> 16

ttgttacaag ctggagaact tctctttgct ggaaccgaag agtcagtatt taatgctgta 60

ttctgtcaaa gaaataaacc acaattgaat ttgatattcg acaaatatga agaaattgtt 120

gggcatccca ttgaaaaagc cattgaaaac gagttttcag gaaatgctaa acaagccatg 180

ttacacctta tccagagcgt aagagatcaa gttgcatatt tggtaaccag gctgcatgat 240

tcaatggcag gcgtcggtac tgacgataga actttaatca gaattgttgt ttcgagatct 300

gaaatcgatc tagaggaaat caaacaatgc tatgaagaaa tctacagtaa aaccttggct 360

gataggatag cggatgacac atctggcgac tannnaaaag ccttattagc cgttgttggt 420

taag 424

<210> 17

<211> 397

<212>DNA

<213> Corn root firefly beetle

<400> 17

agatgttggc tgcatctaga gaattacaca agttcttcca tgattgcaag gatgtactga 60

gcagaatagt ggaaaaacag gtatccatgt ctgatgaatt gggaagggac gcaggagctg 120

tcaatgccct tcaacgcaaa caccagaact tcctccaaga cctacaaaca ctccaatcga 180

acgtccaaca aatccaagaa gaatcagcta aacttcaagc tagctatgcc ggtgatagag 240

ctaaagaaat caccaacagg gagcaggaag tggtagcagc ctgggcagcc ttgcagatcg 300

cttgcgatca gagacacgga aaattgagcg atactggtga tctattcaaa ttctttaact 360

tggtacgaac gttgatgcag tggatggacg aatggac 397

<210> 18

<211> 490

<212>DNA

<213> Corn root firefly beetle

<400> 18

gcagatgaac accagcgaga aaccaagaga tgttagtggt gttgaattgt tgatgaacaa 60

ccatcagaca ctcaaggctg agatcgaagc cagagaagac aactttacgg cttgtatttc 120

tttaggaaag gaattgttga gccgtaatca ctatgctagt gctgatatta aggataaatt 180

ggtcgcgttg acgaatcaaa ggaatgctgt actacagagg tgggaagaaa gatgggagaa 240

cttgcaactc atcctcgagg tataccaatt cgccagagat gcggccgtcg ccgaagcatg 300

gttgatcgca caagaacctt acttgatgag ccaagaacta ggacacacca ttgacgacgt 360

tgaaaacttg ataaagaaac acgaagcgtt cgaaaaatcg gcagcggcgc aagaagagag 420

attcagtgct ttggagagac tgacgacgtt cgaattgaga gaaataaaga ggaaacaaga 480

agctgcccag 490

<210> 19

<211> 330

<212>DNA

<213> Corn root firefly beetle

<400> 19

agtgaaatgt tagcaaatat aacatccaag tttcgtaatt gtacttgctc agttagaaaa 60

tattctgtag tttcactatc ttcaaccgaa aatagaataa atgtagaacc tcgcgaactt 120

gcctttcctc caaaatatca agaacctcga caagtttggt tggagagttt agatacgata 180

gacgacaaaa aattgggtat tcttgagctg catcctgatg tttttgctac taatccaaga 240

atagatatta tacatcaaaa tgttagatgg caaagtttat atagatatgt aagctatgct 300

catacaaagt caagatttga agtgagaggt 330

<210> 20

<211> 320

<212>DNA

<213> Corn root firefly beetle

<400> 20

caaagtcaag atttgaagtg agaggtggag gtcgaaaacc gtggccgcaa aagggattgg 60

gacgtgctcg acatggttca attagaagtc cactttggag aggtggagga gttgttcatg 120

gaccaaaatc tccaacccct catttttaca tgattccatt ctacacccgt ttgctgggtt 180

tgactagcgc actttcagta aaatttgccc aagatgactt gcacgttgtg gtagtctag 240

atctgccaac tgacgaacaa agttatatag aagagctggt caaaagccgc ttttgggggt 300

ccttcttgtt ttatttgtag 320

<210> 21

<211> 47

<212>DNA

<213> Artificial sequence

<220>

<223> Primer YFP-F_T7

<400> 21

ttaatacgac tcactatagg gagacaccat gggctccagc ggcgccc 47

<210> 22

<211> 23

<212>DNA

<213> Artificial sequence

<220>

<223> Primer YFP-R

<400> 22

agatcttgaa ggcgctcttc agg 23

<210> 23

<211> 23

<212>DNA

<213> Artificial sequence

<220>

<223> Primer YFP-F

<400> 23

caccatgggc tccagcggcg ccc 23

<210> 24

<211> 47

<212>DNA

<213> Artificial sequence

<220>

<223> Primer YFP-R_T7

<400> 24

ttaatacgac tcactatagg gagaagatct tgaaggcgct cttcagg 47

<210> 25

<211> 46

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-F1_T7

<400> 25

ttaatacgac tcactatagg gagagctcca acagtggttc cttatc 46

<210> 26

<211> 29

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-R1

<400> 26

ctaataattc ttttttaatg ttcctgagg 29

<210> 27

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-F1

<400> 27

gctccaacag tggttcctta tc 22

<210> 28

<211> 53

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-R1_T7

<400> 28

ttaatacgac tcactatagg gagactaata attctttttt aatgttcctg agg 53

<210> 29

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-F2_T7

<400> 29

ttaatacgac tcactatagg gagattgtta caagctggag aacttctc 48

<210> 30

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-R2

<400> 30

cttaaccaac aacggctaat aagg 24

<210> 31

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-F2

<400> 31

ttgttacaag ctggagaact tctc 24

<210> 32

<211> 48

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Ann-R2T7

<400> 32

ttaatacgac tcactatagg gagacttaac caacaacggc taataagg 48

<210> 33

<211> 47

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-F1_T7

<400> 33

ttaatacgac tcactatagg gagaagatgt tggctgcatc tagagaa 47

<210> 34

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-R1

<400> 34

gtccattcgt ccatccactg ca 22

<210> 35

<211> 23

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-F1

<400> 35

agatgttggc tgcatctaga gaa 23

<210> 36

<211> 46

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-R1_T7

<400> 36

ttaatacgac tcactatagg gagagtccat tcgtccatcc actgca 46

<210> 37

<211> 46

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-F2_T7

<400> 37

ttaatacgac tcactatagg gagagcagat gaacaccagc gagaaa 46

<210> 38

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-R2

<400> 38

ctgggcagct tcttgtttcc tc 22

<210> 39

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-F2

<400> 39

gcagatgaac accagcgaga aa 22

<210> 40

<211> 46

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Betasp2-R2_T7

<400> 40

ttaatacgac tcactatagg gagactgggc agcttcttgt ttcctc 46

<210> 41

<211> 51

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-F1_T7

<400> 41

ttaatacgac tcactatagg gagaagtgaa atgttagcaa atataacatc c 51

<210> 42

<211> 26

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-R1

<400> 42

acctctcact tcaaatcttg actttg 26

<210> 43

<211> 27

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-F1

<400> 43

agtgaaatgt tagcaaatat aacatcc 27

<210> 44

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-R1_T7

<400> 44

ttaatacgac tcactatagg gagaacctct cacttcaaat cttgactttg 50

<210> 45

<211> 50

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-F2_T7

<400> 45

ttaatacgac tcactatagg gagacaaagt caagatttga agtgagaggt 50

<210> 46

<211> 25

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-R2

<400> 46

ctacaaataa aacaagaagg acccc 25

<210> 47

<211> 26

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-F2

<400> 47

caaagtcaag atttgaagtg agaggt 26

<210> 48

<211> 49

<212>DNA

<213> Artificial sequence

<220>

<223> Primer L4-R2_T7

<400> 48

ttaatacgac tcactatagg gagactacaa ataaaacaag aaggacccc 49

<210> 49

<211> 1150

<212>DNA

<213> corn

<400> 49

caacggggca gcactgcact gcactgcaac tgcgaatttc cgtcagcttg gagcggtcca 60

agcgccctgc gaagcaaact acgccgatgg cttcggcggc ggcgtggggag ggtccgacgg 120

ccgcggagct gaagacagcg ggggcggagg tgattcccgg cggcgtgcga gtgaaggggt 180

gggtcatcca gtcccacaaa ggccctatcc tcaacgccgc ctctctgcaa cgctttgaag 240

atgaacttca aacaacacat ttacctgaga tggtttttgg agagagtttc ttgtcacttc 300

aacatacaca aactggcatc aaatttcatt ttaatgcgct tgatgcactc aaggcatgga 360

agaaagaggc actgccacct gttgaggttc ctgctgcagc aaaatggaag ttcagaagta 420

agccttctga ccaggttata cttgactacg actatacatt tacgacacca tattgtggga 480

gtgatgctgt ggttgtgaac tctggcactc cacaaacaag tttagatgga tgcggcactt 540

tgtgttggga ggatactaat gatcggattg acattgttgc cctttcagca aaagaaccca 600

ttcttttcta cgacgaggtt atcttgtatg aagatgagtt agctgacaat ggtatctcat 660

ttcttactgt gcgagtgagg gtaatgccaa ctggttggtt tctgcttttg cgtttttggc 720

ttagagttga tggtgtactg atgaggttga gagacactcg gttacattgc ctgtttggaa 780

acggcgacgg agccaagcca gtggtacttc gtgagtgctg ctggagggaa gcaacatttg 840

ctactttgtc tgcgaaagga tatccttcgg actctgcagc gtacgcggac ccgaacctta 900

ttgcccataa gcttcctatt gtgacgcaga agacccaaaa gctgaaaaat cctacctgac 960

tgacacaaag gcgccctacc gcgtgtacat catgactgtc ctgtcctatc gttgcctttt 1020

gtgtttgcca catgttgtgg atgtacgttt ctatgacgaa acaccatagt ccatttcgcc 1080

tgggccgaac agagatagct gattgtcatg tcacgtttga attagaccat tccttagccc 1140

tttttccccc 1150

<210> 50

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> oligonucleotide T20VN

<220>

<221> misc_feature

<222> (22)..(22)

<223> n is a, c, g or t

<400> 50

tttttttttttttttttttttttttt vn 22

<210> 51

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer P5U76S (F)

<400> 51

ttgtgatgtt ggtggcgtat 20

<210> 52

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer P5U76A (R)

<400> 52

tgttaaataa aaccccaaag atcg 24

<210> 53

<211> 21

<212>DNA

<213> Artificial sequence

<220>

<223> Primer TIPmxF

<400> 53

tgagggtaat gccaactggt t 21

<210> 54

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> Primer TIPmxR

<400> 54

gcaatgtaac cgagtgtctc tcaa 24

<210> 55

<211> 32

<212>DNA

<213> Artificial sequence

<220>

<223> Probe HXTIP

<400> 55

tttttggctt agagttgatg gtgtactgat ga 32

<210> 56

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Spt6v1_360 R

<400> 56

ggaaatacgc aagcagctcg 20

<210> 57

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Probe Spt6v1_224 P

<400> 57

catcctggcg cggtgactcc 20

<210> 58

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Spt6-1v1_62 F

<400> 58

aagatcctgc ctgaacctgc 20

<210> 59

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Spt6-1v2_323 R

<400> 59

ggaaatacgc aagcagctcg 20

<210> 60

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Probe Spt6-1v2_182P

<400> 60

ccaacctccc gaatcgccga 20

<210> 61

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Spt6-1v2_86 F

<400> 61

tcctgcaaga acgtgggtac 20

<210> 62

<211> 151

<212>DNA

<213> Escherichia coli

<400> 62

gaccgtaagg cttgatgaaa caacgcggcg agctttgatc aacgaccttt tggaaacttc 60

ggcttcccct ggagagagcg agattctccg cgctgtagaa gtcaccattg ttgtgcacga 120

cgacatcatt ccgtggcgtt atccagctaa g 151

<210> 63

<211> 69

<212>DNA

<213> Artificial sequence

<220>

<223> Part of the AAD1 coding region

<400> 63

tgttcggttc cctctaccaa gcacagaacc gtcgcttcag caacacctca gtcaaggtga 60

tggatgttg69

<210> 64

<211> 4233

<212>DNA

<213> corn

<400> 64

agcctggtgt ttccggagga gacagacatg atccctgccg ttgctgatcc gacgacgctg 60

gacggcgggg gcgcgcgcag gccgttgctc ccggagacgg accctcgggg gcgtgctgcc 120

gccggcgccg agcagaagcg gccgccggct acgccgaccg ttctcaccgc cgtcgtctcc 180

gccgtgctcc tgctcgtcct cgtggcggtc acagtcctcg cgtcgcagca cgtcgacggg 240

caggctgggg gcgttcccgc gggcgaagat gccgtcgtcg tcgaggtggc cgcctcccgt 300

ggcgtggctg agggcgtgtc ggagaagtcc acggccccgc tcctcggctc cggcgcgctc 360

caggacttct cctggaccaa cgcgatgctg gcgtggcagc gcacggcgtt ccacttccag 420

ccccccaaga actggatgaa cggttagttg gacccgtcgc catcggtgac gacgcgcgga 480

tcgttttttt cttttttcct ctcgttctgg ctctaacttg gttccgcgtt tctgtcacgg 540

acgcctcgtg cacatggcga tacccgatcc gccggccgcg tatatctatc tacctcgacc 600

ggcttctcca gatccgaacg gtaagttgtt ggctccgata cgatcgatca catgtgagct 660

cggcatgctg cttttctgcg cgtgcatgcg gctcctagca ttccacgtcc acgggtcgtg 720

acatcaatgc acgatataat cgtatcggta cagagatatt gtcccatcag ctgctagctt 780

tcgcgtattg atgtcgtgac attttgcacg caggtccgct gtatcacaag ggctggtacc 840

acctcttcta ccagtggaac ccggactccg cggtatgggg caacatcacc tggggccacg 900

ccgtctcgcg cgacctcctc cactggctgc acctaccgct ggccatggtg cccgatcacc 960

cgtacgacgc caacggcgtc tggtccgggt cggcgacgcg cctgcccgac ggccggatcg 1020

tcatgctcta cacgggctcc acggcggagt cgtcggcgca ggtgcagaac ctcgcggagc 1080

cggccgacgc gtccgacccg ctgctgcggg agtgggtcaa gtcggacgcc aacccggtgc 1140

tggtgccgcc gccgggcatc gggccgacgg acttccgcga cccgacgacg gcgtgtcgga 1200

cgccggccgg caacgacacg gcgtggcggg tcgccatcgg gtccaaggac cgggaccacg 1260

cggggctggc gctggtgtac cggacggagg acttcgtgcg gtacgacccg gcgccggcgc 1320

tgatgcacgc cgtgccgggc accggcatgt gggagtgcgt ggacttctac ccggtggccg 1380

cgggatcagg cgccgcggcg ggcagcgggg acgggctgga gacgtccgcg gcgccgggac 1440

ccggggtgaa gcacgtgctc aaggctagcc tcgacgacga caagcacgac tactacgcga 1500

tcggcaccta cgacccggcg acggacacct ggacccccga cagcgcggag gacgacgtcg 1560

ggatcggcct ccggtacgac tatggcaagt actacgcgtc gaagaccttc tacgaccccg 1620

tccttcgccg gcgggtgctc tgggggtggg tcggcgagac cgacagcgag cgcgcggaca 1680

tcctcaaggg ctgggcatcc gtgcaggtac gtctcagggt ttgaggctag catggcttca 1740

atcttgctgg catcgaatca ttaatgggca gatattataa cttgataatc tgggttggtt 1800

gtgtgtggtg gggatggtga cacacgcgcg gtaataatgt agctaagctg gttaaggatg 1860

agtaatgggg ttgcgtataa acgacagctc tgctaccatt acttctgaca cccgattgaa 1920

ggagacaaca gtaggggtag ccggtagggt tcgtcgactt gccttttctt ttttcctttg 1980

ttttgttgtg gatcgtccaa cacaaggaaa ataggatcat ccaacaaaca tggaagtaat 2040

cccgtaaaac atttctcaag gaaccatcta gctagacgag cgtggcatga tccatgcatg 2100

cacaaacact agataggtct ctgcagctgt gatgttcctt tacatatacc accgtccaaa 2160

ctgaatccgg tctgaaaatt gttcaagcag agaggccccg atcctcacac ctgtacacgt 2220

ccctgtacgc gccgtcgtgg tctcccgtga tcctgccccg tcccctccac gcggccacgc 2280

ctgctgcagc gctctgtaca agcgtgcacc acgtgagaat ttccgtctac tcgagcctag 2340

tagttagacg ggaaaacgag aggaagcgca cggtccaagc acaacacttt gcgcgggccc 2400

gtgacttgtc tccggttggc tgagggcgcg cgacagagat gtatggcgcc gcggcgtgtc 2460

ttgtgtcttg tcttgcctat acaccgtagt cagagactgt gtcaaagccg tccaacgaca 2520

atgagctagg aaacgggttg gagagctggg ttcttgcctt gcctcctgtg atgtctttgc 2580

cttgcatagg gggcgcagta tgtagctttg cgttttactt cacgccaaag gatactgctg 2640

atcgtgaatt atttattatta tatatatatc gaatatcgat ttcgtcgctc tcgtggggtt 2700

ttattttcca gactcaaact tttcaaaagg cctgtgtgttt agttcttttc ttccaattga 2760

gtaggcaagg cgtgtgagtg tgaccaacgc atgcatggat atcgtggtag actggtagag 2820

ctgtcgttac cagcgcgatg cttgtatatg tttgcagtat tttcaaatga atgtctcagc 2880

tagcgtacag ttgaccaagt cgacgtggag ggcgcacaac agacctctga cattattcac 2940

ttttttttta ccatgccgtg cacgtgcagt caatccccag gacggtcctc ctggacacga 3000

agacgggcag caacctgctc cagtggccgg tggtggaggt ggagaacctc cggatgagcg 3060

gcaagagctt cgacggcgtc gcgctggacc gcggatccgt cgtgcccctc gacgtcggca 3120

aggcgacgca ggtgacgccg cacgcagcct gctgcagcga acgaactcgc gcgttgccgg 3180

cccgcggcca gctgacttag tttctctggc tgatcgaccg tgtgcctgcg tgcgtgcagt 3240

tggacatcga ggctgtgttc gaggtggacg cgtcggacgc ggcgggcgtc acggaggccg 3300

acgtgacgtt caactgcagc accagcgcag gcgcggcggg ccggggcctg ctcggcccgt 3360

tcggccttct cgtgctggcg gacgacgact tgtccgagca gaccgccgtg tacttctacc 3420

tgctcaaggg cacggacggc agcctccaaa ctttcttctg ccaagacgag ctcaggtatg 3480

tatgttatga cttatgacca tgcatgcatg cgcatttctt agctaggctg tgaagcttct 3540

tgttgagttg tttcacagat gcttaccgtc tgctttgttt cgtatttcga ctaggcatcc 3600

aaggcgaacg atctggttaa gagagtatac gggagcttgg tccctgtgct agatggggag 3660

aatctctcgg tcagaatact ggtaagtttt tacagcgcca gccatgcatg tgttggccag 3720

ccagctgctg gtactttgga cactcgttct tctcgcactg ctcattattg cttctgatct 3780

ggatgcacta caaattgaag gttgaccact ccatcgtgga gagctttgct caaggcggga 3840

ggacgtgcat cacgtcgcga gtgtacccca cacgagccat ctacgactcc gcccgcgtct 3900

tcctcttcaa caacgccaca catgctcacg tcaaagcaaa atccgtcaag atctggcagc 3960

tcaactccgc ctacatccgg ccatatccgg caacgacgac ttctctatga ctaaattaag 4020

tgacggacag ataggcgata ttgcatactt gcatcatgaa ctcatttgta caacagtgat 4080

tgtttaattt atttgctgcc ttccttatcc ttcttgtgaa actatatggt acacacatgt 4140

atcattaggt ctagtagtgt tgttgcaaag acacttagac accagaggtt ccaggagtat 4200

cagagataag gtataagagg gagcaggggag cag 4233

<210> 65

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer GAAD1-F

<400> 65

tgttcggttc cctctaccaa 20

<210> 66

<211> 22

<212>DNA

<213> Artificial sequence

<220>

<223> Primer GAAD1-R

<400> 66

caacatccat caccttgact ga 22

<210> 67

<211> 24

<212>DNA

<213> Artificial sequence

<220>

<223> Probe GAAD1-P (FAM)

<400> 67

cacagaaccg tcgcttcagc aaca 24

<210> 68

<211> 18

<212>DNA

<213> Artificial sequence

<220>

<223> Primer IVR1-F

<400> 68

tggcggacga cgacttgt 18

<210> 69

<211> 19

<212>DNA

<213> Artificial sequence

<220>

<223> Primer IVR1-R

<400> 69

aaagtttgga ggctgccgt 19

<210> 70

<211> 26

<212>DNA

<213> Artificial sequence

<220>

<223> Probe IVR1-P (HEX)

<400> 70

cgagcagacc gccgtgtact tctacc 26

<210> 71

<211> 19

<212>DNA

<213> Artificial sequence

<220>

<223> Primer SPC1A

<400> 71

cttagctgga taacgccac 19

<210> 72

<211> 19

<212>DNA

<213> Artificial sequence

<220>

<223> Primer SPC1S

<400> 72

gaccgtaagg cttgatgaa 19

<210> 73

<211> 21

<212>DNA

<213> Artificial sequence

<220>

<223> Probe TQSPEC (CY5*)

<400> 73

cgagattctc cgcgctgtag a 21

<210> 74

<211> 25

<212>DNA

<213> Artificial sequence

<220>

<223> Primer ST-LS1-F

<400> 74

gtatgtttct gcttctacct ttgat 25

<210> 75

<211> 29

<212>DNA

<213> Artificial sequence

<220>

<223> Primer ST-LS1-R

<400> 75

ccatgttttg gtcatatatt agaaaagtt 29

<210> 76

<211> 34

<212>DNA

<213> Artificial sequence

<220>

<223> Probe ST-LS1-P (FAM)

<400> 76

agtaatatag tatttcaagt atttttttca aaat 34

<210> 77

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Loop_F

<400> 77

ggaacgagct gcttgcgtat 20

<210> 78

<211> 20

<212>DNA

<213> Artificial sequence

<220>

<223> Primer Loop_R

<400> 78

cacggtgcag ctgattgatg 20

<210> 79

<211> 18

<212>DNA

<213> Artificial sequence

<220>

<223> Probe Loop_FAM

<400> 79

tcccttccgt agtcagag 18

<210> 80

<211> 153

<212>DNA

<213> Artificial sequence

<220>

<223> loop linker polynucleotide

<400> 80

agtcatcacg ctggagcgca catataggcc ctccatcaga aagtcattgt gtatatctct 60

catagggaac gagctgcttg cgtatttccc ttccgtagtc agagtcatca atcagctgca 120

ccgtgtcgta aagcgggacg ttcgcaagct cgt 153

<210> 81

<211> 5948

<212> RNA

<213> Corn root firefly beetle

<400> 81

gucacuuguc aauugucaac cuggaaaacg acuugucgaa gucgcauagu uuuuauaagu 60

uuaaauaaac uaaauuaaau auaaauacuu cgagaaugca auaauuauua uucuuuaacu 120

agacccacag cuuauuaauu agcagaagua guagcagacu uauacuaacu agcauaagga 180

gaaacauauu aacauaacau ggcagacuuc auagauucug aagcagaaga aaguagugag 240

gaggaggaau uagaucauag ggaucguaaa aaagcccaaa aagccaaagu uguagauagu 300

ucagaaugaag augaugaaga ugaugacgaa agacugagag aggaauuaaa ggauuugauu 360

gaugauaauc cuauugaaga aagugaugcu gagucugaug cuucaggaag ggaaaaacgu 420

aagaaaucug acgacgagga uuuggaugau cgacuggaag augaagauua ugauuugcuu 480

gaagaaaauu uggguguuaa aguugaaaga aggaaauuca agcgacugcg gcguuuugaa 540

gaugaagaaa gugaaggaga agaagaacau gauccugaac aagauaggga acaaauugcu 600

auggauauau uuucagauga ugacgaugaa agacgaucag aacgaaguca cagaccugcc 660

gucgaacaag aaacuuaugg uguaggcgag gaagaagagg aaggggagua cucggaugcc 720

gaugauuuua uaguugacga ugacgguaga ccgauagcug aaaagaagaa gaagaaaaaa 780

ccaauauuua cugaugccgc ucuccaagag gcucaagaaa ccuucggugu cgauuuugau 840

uaugaugaau uuaguaaaua cgaugaagau gauuacgaag augaagagga gggaggaugac 900

gaauacgagg aagaugaugu agagaaaagg aaacggccua aaaagacuuc aaagaaaaaa 960

ccgacgaaga aauccauuuu ugaaguguau gaaccuagug aacuuaaaag aggguucuuu 1020

accgaucucg auaaugaaau ccgaaacacu gauauucccg aaagaaugca acuucgugau 1080

guuccaauca ccgcuguucc ggaugacuca acugaacuug augaugaagc agaauggauu 1140

uacaggcaag cguuuuguaa cagaacuguu uccaaugugg auucucauuu aucaucagag 1200

gcaagagaga aauuaaagaa gacucaucau gccaucggaa aaaucagaaa agcauuagau 1260

uuuauaagaa aucaacaauu agaaguaccg uuuauugcuu uuuauagaaa ggaguauguu 1320

caaccggaac uuaauauuaa cgauuugugg aaaguauaua aauacgaugc aaaguggugc 1380

caauugaaaa cacgcaaaga aaaccucuug aagcuuuuug aaaaaaugag gucauaucaa 1440

acugaccaca uaaugaaaga uccagaugca ccaauuccag acaaccuucg uauuaugacu 1500

gaguccgaca uugagcgauu gaaaaauguu caaaccgccg aagaguuaaa ugacguucac 1560

aaucauuuca uuuuauauua ugcugcagau uugccagcca ugcaugccgc guggagaguc 1620

aaagaaagag aaaggagaag acaggaaaga aaggaggcua gacuuaagcu caucgcugaa 1680

agugaagaag gugcugaaau uccugaagag ccugaggaaa uugacgauga ugaaccagaa 1740

gcugaaaccu uaaaauacgc caauagauca ggcagcuaug cacuauguaa uaaaggaggu 1800

uuggguccuc uagcgaagaa auuugguua acgccugaag aauuugccga aaaccugaga 1860

gauaauuauc agaggcacga aguagaucaa gaacauauag aaccuguaga agucgcuaaa 1920

gaauucguau cgccgaaauu uccuacagug gaagaagugu ugcaagcuac uaaacacaug 1980

guagcuuuac aaauagcaag agaaccauua guaaggaaau gcguaagaga aauuuucuuu 2040

gaacgagcua gauuaaacgu uuauccaacc aaaaagggug ugaaaguuau agaugaagcu 2100

cauaauugcu auaguaugaa guauguaaaa aauaaaccag uaagagaucu ugcaggcgac 2160

caauuuuuaa aauuauguuu agccgaagag gagaaccucc uuacuauuac caucaaugac 2220

cauauucaag gaaacacuac uaacaauuac auugaugaag ucaaacaauu auauauaaag 2280

gaugaauuca gcaaacacgu ucaggauugg aaugcgcuaa gaauggaauc gguagaaagg 2340

gcuuuaacga aaagugucuu accagauuua agaucugaau uaaaacgaac guugcucaca 2400

gaggcuaaag aauucguauu aaaagcuugc uguagaaaau uauauaauug gauaaagauu 2460

gcuccauacg caauaacuuu ucccgaugaa gacgaagaug auugggauac auccaaaggu 2520

guuagaacua uggguguagc auacguacca gagcacacag uaucagcuuu uaccuguaauu 2580

ucagcaccag acggagauau aacugauuau cucagauuac caaauauucu aaaaagaaaa 2640

aauagcuuuc gaacugaaga aaaacuuaug aaggaagcug aucuucaagc acugaaaaau 2700

uucauauucc uuaaaaagcc ucaugugaua gcaguagggg gugaguccag agaagccuua 2760

augauucgcug augauaucag aggaguaaua agugaacuaa uagaauccga ccaauuccca 2820

cagauuaggg uugaaauuau ugauaaugaa uuagccaaag uguacgcaaa uuccauuaaa 2880

gguucaacug auuucagaga uuauccagag uuguuaaggc aagcuauuuc auuagcucga 2940

agaaugcaag auccuuuggu ugaauuuucc caauuaugua auagcgauga ggaaauuuug 3000

agucugaggu uucaucccuu gcaggaacaa guccagaaag aagaacuacu agaagcucuc 3060

uguuuagaau uugucaacag aacaaaugaa guagguguag auauaaaucu ugccguucag 3120

cagauucaua aaaguaguuu aguucaauuc auaugcgguc uaggaccgcg uaaaggucaa 3180

gcguuacuua aaguucugaa acaaacuaau cagaggcuug aaaacagaac ccaauugguu 3240

acauuuuguc auaugggucc aaaaguuuuu auuaauuguu cuggauucau aaagauugau 3300

accaauaguu uaggagacag uacugaggca uauguugaaa uauuggaugg cucucgaguu 3360

caucccgaaa cuuaugaaug ggcacgaaaa auggcugugg augcuuuaga auaugacgau 3420

gaugaggggag cuaauccggc gggagcuuua gaggaaauuc ucgaggcgcc agagagguua 3480

aaagaaucuug acuuggaugc auuugcggag gaauuggaaa gacaaggauu uggaacaag 3540

aguauaacau uguaugacau uagagcagaa cugaacucgc gauauaaaga uuugagacaa 3600

ccuuuucguu cugcaaaucc ugaagaacua uucgauaugc uuacuaaaga aacucccgaa 3660

acauuuuaua uuggaaaaau gguuacaucu accguguuug gcauugcaag gagaaaacca 3720

aagucagacc agcucgauca agcuaauccg guccguaaug acgaaacugg uuuguggcag 3780

ugccccuucu guuugaaaaa ugauuuuccu gaauuaucug auguauggaa ucauuuugau 3840

gcaggagcau guccugguca agcuacugga guuaaacuaa gacuggauaa ugguauauua 3900

gguuauauuu auauaaaaaa uauaagcgac aaaccaguug cuaauccgga agaaagagua 3960

ggcauaggac aauuaauuca cuguagaaua auaaaaauug acguagagaa guuuaguguu 4020

gauuguacau cgaaaucuag cgaucuugcu gauaaaaauc augaauggag accucaacga 4080

gauccucauu augaucaaga acgugaagac aaagacaauc gauuagaagc cgagaagaag 4140

aaagagaaac agcguaugac guacaucaag aggguuaucg uucauccggc guuccauaac 4200

auauccuaug ccgaagccga aaaauguaug guuaauaugg accaggguga aguuaucauu 4260

aggccuucga guaagggugc ggaucauuug accauuacuu ggaagguuac ggauggaauc 4320

uaucaacaca uugauauuaa ggaacaaggg aaaguuaacg cuuuuucuuu aggaaaauca 4380

uuauggauug gaaaugaaga auuugaagau cuugaugaaa uaauagcuag gcacguuaca 4440

ccaauggcgg cgcaugcuag agauuuacug uauuuuaggu auuacaaaga uuuccaaggu 4500

ggucauaaag auaaggcuga ggaauaucua aaagaugaaa aaaagaaaaa ugcuucuaaa 4560

auucauuaug uaguuagugc ugcuaagaau auaccuggua aauuucuucu uucguaccuu 4620

ccacgcaaca aaguuagaca cgaauaugua acaguuacuc cagaaggcuu ucgguuuaga 4680

caacaaaugu uugauuccgu uucuucguua uuuaaauggu ucaaagaaca uuuccgggag 4740

ccuccaccag gcggugcaac accagguagc acgccaagga uggcuucaag cagaacugga 4800

uauggaagug ccacaccccgc guauaguaug aauaaugaag cuauucaaag aguugcucaa 4860

aauuuaccaa gucauguagu ucaagcguua ucugcugcca ccaaccaaac uccaacauuau 4920

ccccacacccc cugguuaugg aggcaauuau auuaauacgc cuuacacaccc aaguggucaa 4980

acuccauaca ugacuccgua cgcuacaccc cauacgcagc aaacucccccg cuauggucau 5040

caaacaccuu cccaacacau ggcgagcuca gcaccacaag gucucaacaa ucccuuuuua 5100

cauccuggcg cggugacuccc cucccaacga auccuauuu aucgcaacca uccugcacaa 5160

ucuccaguaa ugcuuccuac aagcccugua ccaaguccag guucccagag uucauacagu 5220

agucauuuaa gucauaauca gcgaagugga aguuaugcug aauccuuaag auuccaaccu 5280

cccgaaucgc cgagaagcuc agugaguaau agaagcuuuc aaacugauag auacggcggu 5340

gauagguaug guaaaggagg aagucauaga ugugguggaa gcucaaacga aagauagauau 5400

gguaagggag gaggaggaaa ugaaaauacg gauuggcaga aagcugcaga agcaugggcu 5460

cgaucuagau cuacucccgag aagugauggc cguaauacuc caagaucugu aggucaacga 5520

acuccuagau acgacaacga cgccgaacgu ucuagaauga aacaccucag caagagcccg 5580

aggucuguua ggucuacucc ucgaaccaau acaucucac auucuauguc ccuaggugau 5640

gcuacccccuc uguaugacga aaguaucuaa auuacuuugu auuugguau auaguagggu 5700

aacagugaua uuguaaaugc uaaaaucaac aauacagauu cgcgauauua agucacaaug 5760

acgaccucuc guggaguucg gugaaacuaa cuuugagggc guuuucacug ucaaacgucc 5820

agcgcacaug caccugaaac aaguaggcaa uauucauuau uuauuuuuac agguuuuuua 5880

uacaaauauu auuuacaaca uagacuucaa cuuuuacuga aaggugaagc uuucauauua 5940

auaauuuu 5948

<210> 82

<211> 499

<212> RNA

<213> Corn root firefly beetle

<400> 82

cgccuuacac uccaaguggu caaacuccau acaugacucc guacgcuaca ccccauacgc 60

agcaaacucc ccgcuauggu caucaaacac cuucccaaca cauggcgagc ucagcaccac 120

aaggucucaa caaucccuuu uuacauccug gcgcggugac ucccucccaa cgaacuccua 180

uuuaucgcaa ccauccugca caaucuccag uaaugcuucc uacaagcccu guaccaaguc 240

cagguuccca gaguucauac aguagucauu uaagucauaa ucagcgaagu ggaaguuaug 300

cugaauccuu aagauuccaa ccucccgaau cgccgagaag cucagugagu aauagaagcu 360

uucaaacuga uagauacggc ggugauaggu auguaagg aggaagucau agaauuggug 420

gaagcucaaa cgaagauaga uauguaagg gaggaggagg aaaugaaaau acggauuggc 480

agaaagcugc agaagcaug 499

<210> 83

<211> 145

<212> RNA

<213> Corn root firefly beetle

<400> 83

gacuccguac gcuacaccccc auacgcagca aacucccccgc uauggucauc aaacaccuuc 60

ccaacacaug gcgagcucag caccacaagg ucucaacaau cccuuuuuac auccuggcgc 120

ggugacuccc ucccaacgaa cuccu 145

<210> 84

<211> 108

<212> RNA

<213> Corn root firefly beetle

<400> 84

agaguucaua caguagucau uuaagucaua aucagcgaag uggaaguuau gcugaauccu 60

uaagauucca accucccgaa ucgccgagaa gcucagugag uaauagaa 108

Claims (51)

1. An isolated nucleic acid molecule comprising at least one polynucleotide operably linked to a heterologous promoter, wherein said polynucleotide is selected from the group consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; the complement of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; A native coding sequence of an organism of the genus Chrysophyll, comprising any one of SEQ ID NOs: 3-5; the complement of a native coding sequence of an organism of the genus Chrysophyll, said native coding sequence comprising SEQ ID NO: 3- Any of 5; a fragment of at least 15 contiguous nucleotides of a native coding sequence of an organism of the genus Chrysophyllum comprising any one of SEQ ID NOs: 3-5; and The complement of a fragment of at least 15 contiguous nucleotides of an organism's native coding sequence comprising any one of SEQ ID NOs: 3-5. 2. The nucleic acid molecule according to claim 1, wherein said polynucleotide is selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and any of the foregoing Complementary sequences of those. 3. The nucleic acid molecule according to claim 1, wherein the organism of the genus Chrysophylla is selected from the group consisting of: Osmanthus maizee, Osmanthus barbadensis, Osteophyllum esculentum, Osmanthus mexicani , Cucumber root firefly beetle, cucumber eleven star leaf beetle, South American leaf beetle and D.u.undecimpunctata Mannerheim. 4. The nucleic acid molecule of claim 1, wherein said molecule is a plant transformation vector. 5. A ribonucleic acid (RNA) molecule transcribed from the nucleic acid molecule of claim 1 . 6. A double-stranded ribonucleic acid (dsRNA) molecule expressed from the nucleic acid molecule of claim 1 . 7. The dsRNA molecule according to claim 6, wherein the contact of the polyribonucleotide from the polynucleotide transcribed with the Coleoptera insect inhibits the specific complementary endogenous polyribonucleotide Expression of Derived Nucleotide Sequences. 8. The dsRNA molecule of claim 7, wherein contact of the dsRNA molecule with a Coleopteran insect kills the insect, or inhibits growth, viability and/or feeding of the insect. 9. dsRNA according to claim 6, comprises the first polyribonucleotide, the second polyribonucleotide and the 3rd polyribonucleotide, wherein said first polyribonucleotide Transcribed by said polynucleotide, wherein said third polyribonucleotide is connected to said first polyribonucleotide by said second polyribonucleotide, and wherein said third polyribonucleotide Polyribonucleotide is basically the reverse complementary sequence of described first polyribonucleotide, so that described first polyribonucleotide and described 3rd polyribonucleotide are transcribed into The ribonucleic acid molecules hybridize to form the dsRNA. 10. The RNA of claim 5 selected from the group consisting of double-stranded ribonucleic acid molecules and single-stranded ribonucleic acid molecules of between about 15 nucleotides and about 30 nucleotides in length. 11. The nucleic acid molecule of claim 1, wherein the heterologous promoter is functional in a plant cell. 12. A cell transformed with the polynucleotide of claim 1. 13. The cell of claim 12, wherein the cell is a prokaryotic cell. 14. The cell of claim 12, wherein the cell is a eukaryotic cell. 15. The cell of claim 14, wherein the cell is a plant cell. 16. A plant transformed with the nucleic acid molecule of claim 1. 17. The seed of the plant of claim 16, wherein said seed comprises said polynucleotide. 18. A commercial product produced by the plant of claim 16, wherein said commercial product comprises a detectable amount of said polynucleotide. 19. The plant of claim 16, wherein said at least one polynucleotide is expressed in said plant as a double-stranded ribonucleic acid molecule. 20. The cell of claim 15, wherein the cell is a maize cell. 21. The plant of claim 16, wherein said plant is maize. 22. The plant of claim 16, wherein said at least one polynucleotide is expressed as a ribonucleic acid molecule in said plant, and when a coleopteran insect ingests a part of said plant, said ribonucleic acid molecule Expression of an endogenous polynucleotide that is specifically complementary to the at least one polynucleotide is inhibited. 23. The nucleic acid molecule of claim 1, further comprising at least one additional polynucleotide encoding an RNA molecule that inhibits expression of an endogenous pest gene. 24. The nucleic acid molecule of claim 23, wherein said molecule is a plant transformation vector, and wherein each of said additional polynucleotides is operably linked to a heterologous promoter functional in a plant cell. 25. A method for controlling a population of coleopteran pests, said method comprising providing a medicament comprising a ribonucleic acid (RNA) molecule that acts to inhibit biological activity in said pest after contact with said pest Function, wherein the RNA is specifically hybridizable to a polyribonucleotide selected from the following: any one of SEQ ID NOs:71-74; the complementary sequence of any one of SEQ ID NOs:81-84; A fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; a complementary sequence of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; SEQ ID NO: 81-84 Transcript of any one of ID NO:1 and 3-5; Complementary sequence of transcript of any one of SEQ ID NO:1 and 3-5; At least 15 contiguous nuclei of the transcript of SEQ ID NO:1 A fragment of nucleotides; and the complementary sequence of a fragment of at least 15 contiguous nucleotides of the transcript of SEQ ID NO:1. 26. The method according to claim 25, wherein the RNA of the medicament is specifically hybridizable to a polyribonucleotide selected from the group consisting of: any one of SEQ ID NO:82-84; SEQ ID The complementary sequence of any one of NO:82-84; A fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs:82-84; and at least 15 of any one of SEQ ID NOs:82-84 The complementary sequence of a fragment of contiguous nucleotides. 27. The method of claim 25, wherein the agent is a double-stranded RNA molecule. 28. A method for controlling a coleopteran pest population, said method comprising: Provided is an agent comprising a first polynucleotide sequence and a second polynucleotide sequence, said polynucleotide sequence acting to inhibit a biological function within said coleopteran pest upon contact with said coleopteran pest, wherein said The first polynucleotide sequence comprises a region showing about 90% to about 100% sequence identity to about 15 to about 30 contiguous nucleotides of SEQ ID NO: 81, and wherein the first polynucleotide sequence A nucleotide sequence hybridizes specifically to said second polynucleotide sequence. 29. A method for controlling a coleopteran pest population, said method comprising: In a host plant of a coleopteran pest, a transformed plant cell comprising a nucleic acid molecule according to claim 1 is provided, wherein said polynucleotide is expressed to produce a ribonucleic acid molecule: said ribonucleic acid molecule is related to said ribonucleic acid molecule The contact of a population of coleopteran pests acts to inhibit the expression of a target sequence within said coleopteran pest and to cause said coleopteran pest or population of pests to be relative to a plant of the same host plant species that does not contain said polynucleotide. Reduced growth and/or reduced survival in reproduction of the same pest species. 30. The method of claim 29, wherein the ribonucleic acid molecule is a double-stranded ribonucleic acid molecule. 31. The method of claim 29, wherein the population of coleopteran pests is reduced relative to a population of the same pest species infesting a host plant lacking the transformed plant cells of the same host plant species. 32. The method of claim 30, wherein the population of coleopteran pests is reduced relative to a population of coleopteran pests infesting a host plant of the same species lacking the transformed plant cells. 33. A method of controlling coleopteran pest infestation in plants, said method comprising providing ribonucleic acid (RNA) in the diet of the coleopteran pest, said ribonucleic acid being specifically hybridizable to a polynucleotide selected from the group consisting of: SEQ ID NO:81-84; the complement of any one of SEQ ID NOs: 81-84; A fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; The complement of a fragment of at least 15 contiguous nucleotides of any one of SEQ ID NOs: 81-84; A transcript of any one of SEQ ID NOs: 1 and 3-5; The complementary sequence of the transcript of any one of SEQ ID NO: 1 and 3-5; A fragment of at least 15 contiguous nucleotides of the transcript of SEQ ID NO: 1; and The complement of a fragment of at least 15 contiguous nucleotides of the transcript of SEQ ID NO:1. 34. The method of claim 33, wherein the food material comprises plant cells transformed to express the RNA. 35. The method of claim 33, wherein the specifically hybridizable RNA is contained in a double-stranded RNA molecule. 36. A method for increasing corn crop yield, said method comprising: introducing a nucleic acid molecule according to claim 1 into a corn plant to produce a transgenic corn plant; and Cultivating the corn plant to allow expression of the at least one polynucleotide; wherein expression of the at least one polynucleotide inhibits viability or growth of the coleopteran pest, and yield due to infestation by the coleopteran pest loss. 37. The method of claim 36, wherein expression of said at least one polynucleotide produces an RNA molecule that inhibits at least a first target gene in a coleopteran pest that has contacted a portion of said corn plant . 38. A method for producing a transgenic plant cell, said method comprising: transforming plant cells with a vector comprising a nucleic acid molecule according to claim 1; culturing the transformed plant cells under conditions sufficient to permit development of a plant cell culture comprising a plurality of transformed plant cells; selecting transformed plant cells that have integrated the at least one polynucleotide into their genome; screening said transformed plant cell for expression of a ribonucleic acid (RNA) molecule encoded by said at least one polynucleotide; and Plant cells expressing the RNA molecule are selected. 39. The method of claim 38, wherein the vector comprises a polynucleotide selected from the group consisting of: SEQ ID NO:1; the complement of SEQ ID NO:1; at least 15 contiguous cores of SEQ ID NO:1 A fragment of nucleotides; the complementary sequence of a fragment of at least 15 contiguous nucleotides of SEQ ID NO:1; a native coding sequence of an organism of the genus Chrysophyll, comprising any one of SEQ ID NOs:3-5; leaf The complement of a native coding sequence of an organism of the genus A, said native coding sequence comprising any one of SEQ ID NO: 3-5; a fragment of at least 15 contiguous nucleotides of a native coding sequence of an organism of the genus Chrysophyll, The native coding sequence comprises any one of SEQ ID NOs: 3-5; and the complement of a fragment of at least 15 contiguous nucleotides of a native coding sequence of an organism of the genus Chrysophyll, the native coding sequence comprising SEQ ID NO: Any one of ID NO:3-5. 40. The method of claim 38, wherein the RNA molecule is a double-stranded RNA molecule. 41. A method for producing a transgenic plant protected from damage by a coleopteran pest, the method comprising: providing a transgenic plant cell produced by the method of claim 39; and A transgenic plant is regenerated from said transgenic plant cell, wherein expression of said ribonucleic acid molecule encoded by said at least one polynucleotide is sufficient to regulate expression of a target gene in a Coleopteran pest contacting said transformed plant. 42. A method for producing transgenic plant cells, said method comprising: transforming plant cells with a vector comprising an spt6 construct for providing protection to plants from coleopteran pests; culturing the transformed plant cells under conditions sufficient to permit development of a plant cell culture comprising a plurality of transformed plant cells; selecting transformed plant cells that have integrated into their genome said spt6 construct for providing protection to plants from coleopteran pests; screening said transformed plant cells for expression of an spt6 construct for suppression of expression of essential genes in coleopteran pests; and Plant cells expressing the spt6 component for repression of essential gene expression in coleopteran pests are selected. 43. A method for producing a transgenic plant protected from damage by a coleopteran pest, the method comprising: providing a transgenic plant cell produced by the method of claim 42; and A transgenic plant is regenerated from the transgenic plant cell, wherein expression of the spt6 component for repressing expression of an essential gene in a coleopteran pest is sufficient to regulate expression of a target gene in a coleopteran pest contacting the transformed plant. 44. The nucleic acid of claim 1, further comprising a polynucleotide encoding a polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp. 45. The nucleic acid according to claim 44, wherein the polypeptide from Bacillus thuringiensis is selected from the group consisting of Cry1B, Cry3, Cry34, Cry35, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. 46. The cell of claim 15, wherein the cell comprises a polynucleotide encoding a polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp. 47. The cell according to claim 46, wherein said polypeptide from Bacillus thuringiensis is selected from the group consisting of Cry1B, Cry3, Cry34, Cry35, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. 48. The plant of claim 16, wherein the plant comprises a polynucleotide encoding a polypeptide from Bacillus thuringiensis, Alcaligenes spp., or Pseudomonas spp. 49. The plant of claim 48, wherein said polypeptide from Bacillus thuringiensis is selected from the group consisting of Cry1B, Cry3, Cry34, Cry35, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C. 50. The method of claim 38, wherein the transformed plant cell comprises a nucleotide sequence encoding a polypeptide from Bacillus thuringiensis, Alcaligenes spp. or Pseudomonas spp. . 51. The method according to claim 50, wherein said polypeptide from Bacillus thuringiensis is selected from the group consisting of Cry1B, Cry3, Cry34, Cry35, Cry1I, Cry2A, Cry3, Cry7A, Cry8, Cry9D, Cry14, Cry18, Cry22, Cry23, Cry34, Cry35, Cry36, Cry37, Cry43, Cry55, Cyt1A and Cyt2C.